Cell permeable nanoconjugates of shell-crosslinked knedel (SCK) and peptide nucleic acids (&#34;PNAs&#34;) with uniquely expressed or over-expressed mRNA targeting sequences for early diagnosis and therapy of cancer

ABSTRACT

A functional biologically active particle conjugate useful for diagnosis and treating cancer as a bioportal comprises a nanoscale particle having associated therewith an intracellular targeting ligand comprising a PNA, or another nuclease resistant oligonucleotide analog such as MOE-mRNA (2′-methoxyethyl mRNA) or LNA (locked nucleic acid), having a sequence that binds selectively to an uniquely expressed or overexpressed mRNA specific to the cancer or disease state in a living mammal. In one aspect the uniquely overexpressed target specific to the cancer or disease state is the unr mRNA which can be targeted by the antisense sequence PNA50.

This application claims the benefit of U.S. provisional patentapplication 60/619,242 filed Oct. 15, 2005 which is incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This discovery was made with Government support under Grant 0210247awarded by the National Science Foundation and Contract N01-CO-27103from the National Cancer Institute and NIH-NRSA(5-T32-GM08785-02). TheGovernment has certain rights in this discovery.

FIELD OF THE DISCOVERY

This discovery relates to diagnostic and therapeutic conjugates and useof such conjugates to diagnose and treat cancer. More in particular thisdiscovery relates to biomedical polymer nanoconjugates and their use todiagnose and treat cancer.

BACKGROUND OF THE DISCOVERY

Cancer (malignant neoplasm) is the number two killer of people in theU.S. Each year in the U.S. more than a million people are diagnosed withcancer and half of those will ultimately die from the disease. Canceroccurs when normal living mammalian cells undergo neoplastic (malignant)transformation. Cancer is tenacious in its ability to uncontrollably andrapidly metastasize throughout the mammalian body, thus giving rise to ahigh mortality rate.

Cancer cure rates have increased dramatically over the years. Thispositive trend is a result of the widespread use of improved screeningprocedures that often lead to the early diagnosis/detection of cancer.However, as more selective treatment strategies have been developed, itis necessary to develop new and improved early stage clinical diagnosticprocedures that can be used far earlier to determine a potentialtreatment strategy based on the biological properties and proliferationof the cancer. In addition, it is desired to develop non-invasiveprocedures that can provide the means for determining either a positiveor negative response to a treatment strategy as early as possible thusextending the mammalian host's viability.

The first step in clinically treating cancer is to accurately diagnosethe location and presence of the disease. This means determine thelocation of the cancer and confirm that the suspected cancer is cancer.The area of the body where the tumor or cancer is identified usingsymptomatology reports from a patient and then x-rays or otherdiagnostic tools are utilized to verify the initial symptoms and toidentify the specific location of the cancer. That the location iscancerous is ultimately determined from a biopsy. Examination of asample of suspected cancerous growth by a cancer specialist examiningthe biopsy confirms that the tissue is either benign or it is malignantcancer and if it is cancer, then what type of cancer and what the stageof development of the cancer is determined. Non-invasive imagingtechniques are revolutionizing understanding diseases at the cellularand molecular levels. However, more is needed.

Among the current available non-invasive imaging modalities, positronemission tomography (PET) has demonstrated its great potential in thefield of molecular imaging due to its superior sensitivity andspecificity in diverse applications, the very small amount of agentrequired and the ability to quantitatively analyze the regions ofinterest. Since the completion of human genome sequence, there have beenconsiderable research interests in the assessment of gene functions andprotein-protein interactions non-invasively using molecular imagingapproaches. Of the various PET probes that have been developed to imagegene expression in small animal models, oligonucleotides appear to be aninexhaustible gold mine for the development of new tracers with highspecificity considering that an oligonucleotide with more than 12nucleobases could represent a unique sequence in the whole human genomewith a gene number estimated between 24,000 and 30,000, and alternativepolyadenylation and splicing could result in a number of messengerribonucleic acid (mRNA) between 46,000 and 85,000.

The techniques of antisense mRNA originated from the natural modulationof gene replication and expression in bacteria via small complementaryRNA molecules in an opposite direction (antisense). However, thenaturally occurring oligonucleotides with 2′-deoxyphosphodiester cannotbe directly applied to nuclear imaging because they are rapidly degradedin vivo by endo- and exo-nucleases. To increase the in vivo stability ofoligonucleotides without significant alteration of theirpharmacokinetics and targeting properties, many chemical modificationshave been made to the sugar-phosphate backbone, including morpholino,phosphorothioate, phosphoroamidate, methylphosphonate, 2′- or3′-modified derivatives, peptide nucleic acids (PNAs), and lockednucleic acids. Peptide nucleic acids (PNAs) are a unique type ofoligonucleotides, which was initially introduced by Nielsen et al. in1991 as ligands for the double stranded DNA recognition. They aresynthetic DNA mimics featuring a chain with repeating N-(2-aminoethyl)glycine units instead of the sugar-phosphate backbone.

Due to the structural characteristics (e.g., neutrality andflexibility), PNAs are resistant to the in vivo enzymatic degradation.PNAs bind complementary DNAs or RNAs with high affinity and specificityeven under low ionic strength conditions. Recent work has shown thatPNAs can be used as molecular hybridization probes, and nuclear imagingtracers.

Using antisense PNAs as molecular imaging probes has a major obstacle inthat they have very poor permeability across biologic membranes, whichis inherent from their structural feature. Therefore for thehybridization of an unmodified PNA with its target mRNA molecule invitro, it often requires that the PNA be physically injected into theintracellular plasma. Attempts to overcome this obstacle have beenresorted to the drug-delivery techniques, which include using cationiclipids (or polyamines) and liposomes, nanoparticles, and directconjugation with monoclonal antibody or peptides, etc. Recently it wasreported that PNAs with four lysines at the C terminus (PNA-K4oligomers) demonstrated sequence-specific antisense activity in mosttissues that expressed a specific gene.

Radionuclides such as ⁶⁰Cu, ⁶¹Cu and ⁶⁴Cu among other radionuclidesrespectively are utilized extensively in the diagnosis and treatment ofcancer in living mammals. These radionuclides are useful for diagnosis(⁶⁰Cu, ⁶¹Cu and ⁶⁴Cu); internal radiation therapy (⁶¹Cu and ⁶⁴Cu)because of their positron-emission and/or toxicity to cancer and theircharacteristic intermediate half-life and multiple decay mode. Suchdiagnostic and therapeutic efforts against cancer include the effectiveadministration of radiolabeled chemicals using highly purified ⁶⁰Cu,⁶¹Cu and ⁶⁴Cu. ⁶⁴Cu is especially useful. The principal advantage tosuch use is that the radionuclide identifies a location for the canceras well as provides a cytotoxic effect against the cancer.

Synthetic methodologies are enabling advances in the design of polymericmaterials that actively control cellular and physiologic responses.These methods produce materials that are incorporated into scaffoldsadaptive to body blood lumens and capable of performing specificfunctions while being minimally detrimental to normal cellular processesand surrounding tissues for use in therapeutic, drug delivery, andtissue engineering applications.

However, a need continues to exist for enhanced methods that can moreaccurately diagnose cancer and more particularly assess the response toanticancer therapy, as such methods would have a significant positiveimpact on determining optimal therapy for treating cancer patients. Alsonew methods are needed to treat cancer.

So despite the aforegoing remarkable advances and other advances incancer diagnostics and cancer detection technology, it remains highlydesirable to have an enhanced cancer detection and treatment system foruse in a living mammal such as in a living human.

BRIEF DESCRIPTION OF THE DISCOVERY

This discovery provides a functional biologically active functionalwater dispersible synthetic conjugate useful for early stage diagnosingand treating cancer in a living mammal, comprising a nanoscale particlehaving associated therewith an intracellular targeting ligand comprisinga PNA, or another nuclease resistant oligonucleotide analog such asMOE-mRNA (2′-methoxyethyl mRNA) or LNA (locked nucleic acid), having asequence that binds selectively to an uniquely expressed oroverexpressed mRNA specific to the cancer or disease state in a livingmammal. In one aspect the uniquely overexpressed target specific to thecancer or disease state is the unr mRNA (see SEQ ID 2) which can betargeted by the antisense sequence PNA50 (see SEQ ID 3).

In a further aspect, a permeation peptide is additionally associatedwith a biologically active particle conjugate and PNA. In a furtheraspect the permeation peptide is an effective permeation peptide whichcomprises the HIV-1 TAT protein transduction domain further comprising apolypeptide having a sequence such as the sequence shown in SEQ ID 1.

In a further aspect, a functional biologically active particle conjugatecomprises water dispersible biomedically and pharmacologicallyacceptable polydispersity globular macromolecules, particles, ornanoparticles. Useful particles include those particles disclosed in theU.S. Pat. No. 6,383,500 which issued to Karen Wooley et al. on May 7,2002 (hereafter referred to as the '500 patent) which is incorporatedherein in its entirety by reference. Further useful particles includedendrimers, micelles, liposomes, etc. as disclosed in Pharmacol. Rev.2001, 53, 283-318; Nature Rev. Drug Disc. 2003, 2, 347-360.

In an aspect a method of identifying uniquely expressed or overexpressedmRNA comprises using SAGE or DNA chip to quantify gene expression in atarget cell, comparing the gene expression profile to expressiondatabases, and identifying a sequence that is most differentiallyexpressed and is in the highest amount or is uniquely expressed toidentify an mRNA of interest, obtaining a clone containing the cDNA forthe mRNA of interest and producing the mRNA in vitro by RNA polymerase,mapping accessible sites by either the modified RT-ROL assay and/orSAABS assay, screening potential ODNs by the Dynabead dot blot assay andquantifying the binding of ODNs by the Dynabead direct binding assaywith ³²P-labeled ODN.

In a further aspect, the identified sequence is modified to obtain auseful targeting ligand by synthesizing and recovering Cys-Tyr-PNA-Lys4corresponding to the tightest binding ODNs (or with another permeationpeptide in place of Lys4), quantifying binding of the hybrid PNAs by theDynabead direct binding assay with radioiodinated PNA, conjoining thehighest affinity PNAs to fluorescein and DOTA for fluorescence assays ofcell binding in vitro or in vivo (mouse xenograft) and conjoining PNAswith the highest affinity to SCK nanoparticles through an appendedlysine or other suitable accommodating site-specific coupling moiety.

In an aspect, an isolated, purified and characterized PNA having a unrmRNA binding antisense sequence PNA50 (see SEQ ID 3).

In an aspect, an intracellular targeting ligand comprises a PNA, oranother nuclease resistant oligonucleotide analog such as MOE-mRNA orLNA, having a sequence that binds selectively to an uniquely expressedor overexpressed mRNA specific to the cancer or disease state and apermeation peptide (e.g., HIV-1 TAT protein transduction domain, See SEQID. 1). In one aspect the uniquely overexpressed target specific to thecancer or disease state is the unr mRNA (see SEQ ID 2) which can betargeted by the antisense sequence PNA50 (See SEQ ID 3).

In an aspect, an intra intracellular targeting ligand useful fordetection of cancer in a living mammal comprises a PNA, or anothernuclease resistant oligonucleotide analog such as MOE-mRNA or LNA,having a sequence that binds selectively to an uniquely expressed oroverexpressed mRNA specific to the cancer or a nuclease resistantoligonucleotide analog MOE-mRNA or LNA, a permeation peptide and areporter capable of detecting cancer, such as an emission capablefluorophore or a radionuclide or both a fluorophore and a radionuclide.In an aspect the living mammal is a living human.

In an aspect, an active targeting cancer detection system comprises aparticle based moiety having associated therewith a PNA or anothernuclease resistant oligonucleotide analog that does not activate thedegradation of the mRNA by RNase H, such as MOE-mRNA or LNA, having aunr mRNA binding sequence such as PNA50 (See SEQ ID 3) or any sequencethat binds selectively to an unique or overexpressed mRNA specific tothe cancer or disease state, a permeation peptide (e.g., HIV-1 TATprotein transduction domain See SEQ ID 1), and a reporter capable ofdetecting cancer, such as an emission capable fluorophore or aradionuclide or both a fluorophore and a radionuclide.

In an aspect, a diagnostic target-specific imaging probe comprises aparticle-based moiety having associated therewith a PNA or anothernuclease resistant oligonucleotide analog that does not activate thedegradation of the mRNA by RNase H, such as MOE-mRNA or LNA, having aunr mRNA binding sequence such as PNA50 (see SEQ ID 3) or any sequencethat binds selectively to an uniquely expressed or overexpressed mRNAspecific to the cancer or disease state, a permeation peptide (e.g.,HIV-1 TAT protein transduction domain, see SEQ ID 1), and a diagnosticimaging detectable amount of at least one detectably labeled compound.

In an aspect, a method of detecting cancer comprises administering aneffective amount of a particle-based moiety having associated therewitha PNA or another nuclease resistant oligonucleotide analog that does notactivate the degradation of the mRNA by RNase H, such as MOE-mRNA orLNA, having a unr mRNA binding sequence such as PNA50 (see SEQ ID 3) orany sequence that reactively and selectively binds to an uniquelyexpressed or overexpressed mRNA specific to the cancer, and a permeationpeptide and a reporter capable of detecting the cancer, such as anemission-capable fluorophore, or a radionuclide, or both a fluorophoreand a radionuclide. In an aspect the detected reporter identifies thelocus of the cancer in the living mammal.

In an aspect, an anticancer composition effective for treating human ornon-human neoplastic disorders comprises a particle-based moiety havingassociated therewith at least a PNA or another nuclease resistantoligonucleotide analog that does not activate the degradation of themRNA by RNAseH, such as MOE-mRNA or LNA, having a unr mRNA bindingsequence such as PNA50 (see SEQ ID 3) or any sequence that bindsselectively to an uniquely expressed or overexpressed mRNA specific tothe cancer or disease state, a permeation peptide, and an effectiveamount of at least one chemotherapeutic compound, cytotoxic compound,radionuclide or prodrug in the composition, and optionally furthercomprising a pharmaceutically acceptable carrier such as salinesolution.

In an aspect, a radiotherapeutic composition effective for treatinghuman or non-human neoplastic disorders comprises a particle conjugatecomprising a particle-based moiety having associated therewith at leasta PNA or another nuclease resistant oligonucleotide analog that does notactivate the degradation of the mRNA by RNase H, such as MOE-mRNA orLNA, having a unr mRNA binding sequence such as PNA50 (see SEQ ID 3) orany sequence that binds selectively to an uniquely expressed oroverexpressed mRNA specific to the cancer or disease state, a permeationpeptide, and an effective amount of at least one radionuclide withcytotoxic properties, and optionally further comprising apharmaceutically acceptable carrier such as saline solution.

In an aspect, a method for determining response to anticancer therapy ina living mammal comprises administering to a living mammal an imagingprobe comprising a particle-based moiety having associated therewith aPNA or another nuclease resistant oligonucleotide analog that does notactivate the degradation of the target mRNA by RNase H, such as MOE-mRNAor LNA, having a unr mRNA binding sequence such as PNA50 (see SEQ ID 3)or any sequence that binds selectively to a unique or overexpressed mRNAspecific to the cancer or disease state, a permeation peptide, and adiagnostic imaging detectable amount of at least one detectably labeledcompound at a first selected time, detecting an image of a tissue,administering the imaging probe a second time after the anticancertherapy, detecting an image of the same tissue, comparing the two imagesand determining a response based on that comparison.

In an aspect, a method of screening candidate chemicals fortoxicity/lethality to cancer comprises administering, to a mammal, aparticle conjugate comprising a PNA or another nuclease resistantoligonucleotide analog that does not activate the degradation of themRNA by RNase H, such as MOE-mRNA or LNA, having a unr mRNA bindingsequence such as PNA50 (see SEQ ID 3) or any sequence that bindsselectively to a uniquely or overexpressed mRNA specific to the canceror disease state, a permeation peptide, and diagnostic imagingdetectable amount of at least one detectably labeled compound at a firsttime, detecting and acquiring an image of a tissue, administering to themammal a candidate chemical, detecting and acquiring an image of tissue,comparing the detected images and making a determination as to whetherthere has been a prophylactic effect on the progression of the cancer.In an aspect the PNA is conjugated to a biologically active waterdispersible nanoparticle.

In as aspect, a pharmaceutical kit comprises a container. In an aspectthe container houses a PNA or another nuclease resistant oligonucleotideanalog that does not activate the degradation of the target mRNA byRNase H, such as MOE-mRNA or LNA, having a unr mRNA binding sequencesuch as PNA50 (see SEQ ID 3) or any sequence that binds selectively toan unique or overexpressed mRNA specific to a cancer or disease state, apermeation peptide, a pharmaceutical agent selected from the groupconsisting of chemotherapeutic drugs, cytotoxic drugs, prodrugs, and aradiopharmaceutical, and optionally a suitable pharmaceuticallyacceptable carrier. In an aspect the PNA is conjugated to a biologicallyactive nanoparticle.

In an aspect, a PNA-guided tumor therapy comprises administering to amammalian patient a therapeutically effective amount of a PNA or anothernuclease resistant oligonucleotide analog that does not activate thedegradation of the mRNA by RNase H, such as MOE-mRNA or LNA, having aunr mRNA binding sequence such as PNA50 (see SEQ ID 3) or any sequencethat binds selectively to a unique or overexpressed mRNA specific to thecancer or disease state and a permeation peptide, whereby the particleconjugate is self guided to the tumor by the PNA and a pharmaceuticalagent selected from at least one chemotherapeutic drug, cytotoxic drug,prodrug and a radiopharmaceutical. In an aspect the PNA is conjugated toa biologically active nanoparticle.

In an aspect, a medical apparatus useful for treating and detectingcancer, comprises a particle based moiety functioning as a scaffold andhaving associated therewith a PNA or another nuclease resistantoligonucleotide analog that does not activate the degradation of themRNA by RNase H, such as MOE-mRNA or LNA, having a unr mRNA bindingsequence such as PNA50 (see SEQ ID 3) or any sequence that bindsselectively to a unique or overexpressed mRNA specific to cancer ordisease state, and a permeation peptide. In an aspect the unique oroverexpressed mRNA is a signature of cancer or a disease state in aliving mammal and the methods, compositions, apparatus and scaffoldingdescribed herein provide a method of uniquely diagnosing and targetingthat signature.

In an aspect, a bioactive pharmaceutical composition useful for treatingcancer comprises a PNA or another nuclease resistant oligonucleotideanalog that does not activate the degradation of the mRNA by RNase H,such as MOE-mRNA or LNA, having a unr mRNA binding sequence such asPNA50 (see SEQ ID 3) or any sequence that binds selectively to auniquely expressed or overexpressed mRNA specific to the cancer ordisease state, and a permeation peptide. If desired, the pharmaceuticalcomposition comprises a pharmaceutical, or a pharmaceutically acceptablewater soluble salt thereof, and a pharmaceutically acceptable carrier,excipient, or diluent or saline composition. The pharmaceutically activeagent can be present within the particles. In an aspect a nano-scaleparticle-based moiety is conjugated to the PNA or analog.

In a further aspect, a method of effectively delivering a bioactivepharmaceutically active agent to a cell, tissue, organ, or animal,comprises contacting the cell, tissue, organ, or animal in vivo or invitro, with a PNA or another nuclease resistant oligonucleotide analogthat does not activate the degradation of the mRNA by RNase H, such asMOE-mRNA or LNA, having a unr mRNA binding sequence such as PNA50 (seeSEQ ID 3) or any sequence that binds selectively to a unique oroverexpressed mRNA specific to the cancer or disease state, a permeationpeptide and further comprising a pharmaceutically active agent. Inaspect the permeation peptide comprises HIV-1 TAT protein transductiondomain having SEQ ID. 1). In an aspect a biologically active particle isassociated with the PNA or analog.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting some aspects of the invention: theparticle-based unr mRNA targeting moiety DOTA-SCK(PTD)-PNA50 andnon-targeting control DOTA-SCK(PTD)-PNA50S, and the unr mRNA targetingmoiety DOTA-PNA50-K4 and non-targeting control DOTA-PNA50S-K4

FIG. 2 depicts the synthesis of a SCK-PNA-FTSC-PTD construct.

FIG. 3 depicts the synthesis of a SCK-PNA-PTD-DOTA construct.

FIG. 4 shows UV visible spectra of SCK and recovered SCK-PNA conjugatesshowing a graph of absorbance data as a function of wavelength for SCK,SCK-PNA50 and SCK-PNA50S.

FIG. 5(A) and FIG. 5(B) show inventor generated data of sedimentationvelocity analysis FIG. 5(A) (left side) shows data from the result of asedimentation velocity analysis of SCK-PNA50 conjugate. The loss ofabsorbance at 260 nm at the top of the cell (near the meniscus)demonstrated the desired absence of free PNA within the reactionsolution. The data is data of intensity as a function of radius. FIG.5(B) (right side) shows a detail of UV spectrum recorded near themeniscus.

FIG. 6 depicts schematically the structure of DOTA-NH₂-tri-tert-butylester, the chelator used for effective conjugation to the SCK-PNA-PTDconstruct.

FIG. 7 shows that the inventors' conjugation of a PTD sequence to SCKhas desired positive dramatic effects on resulting cell viability. The2.0% PTD-functionalized SCK 9 is toxic to the cells between 7 mol/L to13 mol/L in PTD. The 0.5% and 1.0% PTD-functionalized SCKs (7 and 8) aretoxic between 14 mol/L to 28 and 7 mol/L to 15 mol/L in PTD,respectively. The parent micelles and SCKs (5 and 6) did not affect theviability of the cells at any measured concentration. Error bars arerepresentative of one standard deviation from the mean of quadruplicatesamples harvested form four separate wells of mouse myeloma B cells andare estimates of the standard uncertainties.

FIG. 8 shows four distinct populations of cells identified with flowcytometry (viable, early apoptotic, late apoptotic and necrotic) more indetail. Viable cells: Annexis V(−) and 7-AAD(−); early apoptotic;Annexin V (+) and 7-AAD(−); late state apoptotic: Annexin V (+) and7-ADD (+) necrotic; Annexin V (−) and 7-ADD (+). Error bars arerepresentative of one standard deviation from the mean of triplicatesamples, each harvested from three separate populations of RAW 264.7cells and the estimates are of the standard uncertainties.

FIG. 9 shows a plot of inventors' data from genetic cellularinflammatory responses exhibited 24 hours after incubation withdifferent concentrations of peptide functionalized SCK nanoparticlesquantified by RT-PCR. These data depict minimal increases in theproduction of TNF-α and an elevation in the levels of IL-1 relative totissue culture deviation from the mean of triplicate samples, eachharvested from three separate populations of RAW 264.7 cells, and theestimates are of the standard uncertainties.

FIG. 10 shows each group of three bars within a data set correspondingto a titer measurement from an individual living mouse. The red barscorrespond to the prebled, the orange bars represent the bleed two weeksafter the first booster immunization, and the yellow bars represent thebleed two weeks following the end booster immunization. Detrimentalimmunogenic effects, relative to controls, are negligible in all samplesexcept for the 2.0% (9) sample which suggests that the quantity of PTDand the manner in which it is presented on the surface have asynergistic effect that is able to produce an immune responsesignificantly greater than the response elicited by the peptide itself.Error bars are representative of one standard deviation from the mean oftriplicate samples harvested from two separate serum vials and theestimates are of the standard uncertainties.

FIG. 11 shows that sequential ATRP of tert-butyl and methyl acrylateafforded well-defined poly (tert-butyl acrylate-b-methyl acrylate). Thetert-butyl esters were successfully cleaved selectively throughtreatment of the diblock copolymer with TFA in CH₂Cl₂ for 36 hours.

FIG. 12 shows that SCK (denoted as 6 in FIG. 12) was prepared throughthe micellization of the block copolymer 4, followed by the crosslinking of approximately 50% acrylic acid residues in the shell layerwith 2,2′ (ethylenedioxy bis(ethylamine)) The SCKs (denoted as 7-9 inFIG. 12) were functionalized with various molar quantities of PTD underhigh ionic strength conditions to minimize aggregation and interparticlecross linking. A linker derivatized 5(6)-carboxyfluorescein dye(approximately 70 fluorophores per particle) was coupled to each of theSCK samples 6-9 to facilitate observation under fluorescence conditions.

FIG. 13 shows the sedimentation equilibrium profiles the inventorscollected using an interferometry detector for the micelles, SCKs andpeptide-functionalized nanoparticles. In FIG. 13(A) the dramaticincrease in weight average molecular weight (Mw) was due to a smallpercentage of interparticle cross-linking (covalent or electostatic),which contributed significantly to the molecular weight calculations. InFIG. 13(B), UV spectra recorded near the meniscus of solutions atsedimentation equilibrium showed negligible absorbance due to freetyrosine residues within the PTD relative to controls. In FIG. 13(C)data show the repeating of the sedimentation test at 8000 rpm andindicated that the meniscus was fully depleted of SCK at 8000 rpm, butnot at 5000 rpm, and also confirmed that all the PTD was desirablyattached to the SCK.

FIG. 14 shows UV visible spectroscopy which afforded the quantitativecalculation of the number of peptides per particle by two differentmethods. The first method involved the simultaneous solving of twounknowns as determined from PTD (FIG. 14(A)) and SCK (FIG. 14(B))concentration calibration plots at 230 nm and 276 nm. From thecalibration curves generated from FIG. 14(A) and FIG. 14(B), theconcentration of PTD in the respective SCK solutions shown in FIG. 14(C)were calculated. The large number of peptides calculated to be in the2.0% sample is believed to be a result of the slight amount of turbiditypresent in that sample due to aggregation which is reflected in theabsorbance measurement of that sample (orange). The second methodinvolves the measurement of phenylglyoxal derivative formation FIG.14(D). Phenylglyoxal reacts specifically with the guanidine groups onthe side chains of arginine residues yielding a derivative with anabsorbance red shifted (310 nm) from that of the parent compound. From acalibration curve generated from known amounts of PTD, the molarconcentration of the peptide in unknown solutions was determined. Molarratios were then applied to quantify the number of peptides perparticle.

FIG. 15 depicts live cell confocal fluorescence microscopy showinginternalization of the 0.5, 1.0 and 2.0% PTD-functionalizednanoparticles at both 37° C. and 4° C. in CHO cells. No uptake orinternalization was seen for the unfunctionalized parent SCK under livecell conditions.

FIG. 16 shows confocal fluorescence microscopy of PTD-functionalizedSCKs using CHO cells fixed with a 4% paraformaldehyde solution. It showsnonspecific uptake of the nonfunctionalized SCKs which was not observedin live cell tests, in addition to the qualitatively enhanced uptake ineach of the PTD functionalized samples. FIG. 16(A) and FIG. 16(B) showconfocal reconstructions of the respective SCK samples at 37° C. and 4°C. respectively, following 1 hour incubations and cell fixation. FIG.16(C) shows CHO cells that were incubated for 1 hour in 0.1% sodiumazide with the SCK samples.

FIG. 17 is an inventors' schematic depicting antisense cellulartargeting of prodrugs and probes. FIG. 17 depicts an antisense agentsynthesized against an abundant unique or overexpressed mRNA andconjoined to a probe (or prodrug) and permeation peptide, directly orvia an attached nanoparticle. The permeation peptide allows the hybridPNA to equilibrate with other cells and ultimately concentrate in thecell containing the more abundant mRNA.

FIG. 18 depicts a modified RT-ROL assay. In this assay antisense bindingsites are identified by the ability of a random 9-mer oligonucleotidelibrary (ROL) terminating a specific base and a PCR tag to primecomplementary DNA (cDNA) synthesis by reverse transcriptase (RT). ThecDNA is then amplified by PCR with a radiolabeled primer having the samesense as the RNA, and an unlabelled primer having the same sequence asthe PCR tag.

FIG. 19 shows in 19(A) primers used to map ODN accessible sites on unrtranscript. FIG. 19 shows the location of unrX (X=1-7) priming sites.FIG. 19(B) shows phosphorimages of acrylamide gels of PCR products foreach section (X=1-7) of the unr transcript. The boxed site of FIG. 19 isanalyzed in FIG. 20. Primer sequences: unr1: GCTGAGCTGTTGGGTATGAAG,unr2: TCATCCTTTGAAACGTGTGC, unr3: ACGAACGTAATGGGGAAGTG; unr4:AAATCCAAGGTGACCCTGCT; unr5: TGACTGTGGGGTGAAACTGA; unr6:GAGGGCGATATGAAAGGTGA unr7: AACCACATCCACAAAGCACA.

FIG. 20 shows interpretation of the RT-ROL gel. FIG. 20(A) is anenlargement of boxed section of gel in FIG. 19 showing approximate sizeof bands. FIG. 20(B) shows alignment of the observed bands with the mRNAsequence, taking into account that the PCR tag adds an additional 20nucleotides to the extra 9 random nucleotides in the PCR amplifiedproduct band.

FIG. 21 depicts the results of an SAABS assay. A random 8-meroligodeoxynucleotide library (ROL) flanked by two PCR tags is incubatedwith mRNA bound to a Dynabead through hybridization of the attachedcRNA-Bio, and then separated from the unbound sequence by a magneticfield. The bound sequence is then PCR amplified, restricted with NlaIIIconcatenated by ligation, cloned in pZEro and sequenced as shown in FIG.21. Further, the sequences of the ODNs are as follows: S1-ROL-S2:GGATTTGCTGGTGCAACATGN₈CATGAAGCTTGA AATTCGAGG; S1: GGATTTGCTGGTGCAACATG;CS1: CATGTTGCACCAGCAAATCC; CS2: CCTCGAATTCAAGCTTCATG;CRNA-Bio:Biotin-TGGTCCTCAGAATTAAAGCATAATG.

FIG. 22 shows frequency distribution of the antisense binding sequencesobtained from the SAABS assay. The 8-mer sequences were retrieved fromthe sequenced clones and aligned with the mRNA sequence. Some of thesites identified correspond to sites found by the RT-ROL assay (13 and46), whereas others were uniquely detected by the SAABS assay anddenoted with an S prefix (S1, S3, S5 and S7).

FIG. 23 shows the results of a Dynabead-based dot blot assay todetermine relative binding affinity of ODNs. FIG. 23(A) Determining theloading capacity of the streptavidin coated dynabead by titrating 20 μLof bead solution in 40 μL total volume of 0.5 M NaCl with biotinylatedradiolabeled unr mRNA. FIG. 23(B) shows a determination of the μL ofDynabead bound RNA needed to completely bind 1 pmol of ODN5 in a totalvolume of 40 μL. FIG. 23(C-D) Solutions of RNA were incubated with 1pmol of ODN (1-54 from RT-ROL assay, and 57-68 (S1-S12) from the SAABSassay) and then incubated with 10 μL of Dynabeads and spotted on a Nylonmembrane. FIG. 23(C) is a photograph of blot showing equal loading ofbeads. Panel D is a radiograph showing relative amounts of retained ODN.

FIG. 24 is an inventors' schematic depicting the results of aDynabead-based quantitative binding assay. The solution containing freeODN is physically separated from the ODN bound to the RNA by a magneticfield and is quantified by liquid scintillation counting.

FIG. 25 provides inventors' data curve fits of the data from theDynabead ODN binding assay depicted in FIG. 24.

FIG. 26 shows design and radioiodination of PNAs with ¹³¹I. PNAscontained a tyrosine which can be readily iodinated by I₂ under neutralconditions. The ¹³¹I₂ was successfully produced in situ by oxidationwith chloramine-T.

FIG. 27 shows successful iodination reactions including HPLC analysis ofiodination reaction with chloramine-T (b & c) and IODO-beads (d & e) forthe times shown. Products were identified by MALDI-TOF.

FIG. 28 shows inventors' data curve fits to the PNA binding data fromthe Dynabead assay. The error bars represent the standard deviation ofthe average of three tests.

FIG. 29 shows RT-PCR assay for demonstrated successful binding of PNA50to unr mRNA isolated from MCF-7 cells. A) dT18, B) ODN50, C) PNA50, D)PNA50+ODN50, E) PNA50+ODN50+dT18, F) PNA50S+ODN50, G)PNA50S+ODN50+dT18H) no PNA or ODN. (RT is reverse transcriptase, PCR wascarried out with Taq polymerase.)

FIG. 30 shows fluorescence microscopy of MCF-7 cells followingincubation with the PNAs shown for the times shown.

FIG. 31 shows fluorescence of MCF-7 cell lysates following 24 hourincubation with fluorescein labeled PNAs. Bars 1-4 show the PNAsfluorescein coupled to their amino terminus and the NLS peptide sequenceto their carboxyterminus. B shows a blank control with no fluorescentlylabeled PNA added. C shows fluorescein labeled NLS sequence without aPNA attached.

FIG. 32 shows biodistribution data of ⁶⁴Cu-DOTA-PNA-K4 conjugates inselected organs of normal balb/c mice via the tail vein injection.Injection dose: 10-12 μCi/100 μL. FIG. 32(A) (top): ⁶⁴Cu-DOTA-PNA50-K4;FIG. 32(B) (bottom): ⁶⁴Cu-DOTA-PNA50S-K4. Data are presented as percentinjected dose per organ (% ID/organ). Error bars are representative ofone standard deviation from the mean of three living animals.

FIG. 33 shows biodistribution data of ⁶⁴Cu-DOTA-PNA-K4 conjugates inselected organs of normal balb/c mice via intraperitoneal injection.Injection dose: 55 μCi/100 μL. FIG. 33(A) (top): ⁶⁴Cu-DOTA-PNA50-K4;FIG. 33(B) (bottom): ⁶⁴Cu-DOTA-PNA50S-K4. Data are presented as percentinjected dose per organ (% ID/organ). Error bars are representative ofone standard deviation from the mean of three living animals.

FIG. 34 shows MicroPET transaxial images of ⁶⁴Cu-labeled PNA conjugatesin MCF-7 tumor bearing CF-17 SCID mice. FIG. 34(a) ⁶⁴Cu-DOTA-PNA50-K4(injection dose: 210 μCi); FIG. 34(b) ⁶⁴Cu-DOTA-PNA50S-K4 (injectiondose: 347 μCi); FIG. 34(c) ⁶⁴Cu-DOTA-PNA5-K4 (injection dose: 253 μCi);and FIG. 34(d) ⁶⁴Cu-DOTA-PNA7-K4 (injection dose: 361 μCi). Theintensity of the images was decay-corrected and normalized to theinjection dose.

FIG. 35 shows MicroPET coronal image of ⁶⁴Cu-DOTA-PNA50-K4 in a MCF-7tumor bearing CF-17 SCID mouse at 1 h p.i. The intensity of the imageswas scaled by max/min of frame.

FIG. 36 shows time-activity curves of ⁶⁴Cu-DOTA-PNA-K4 conjugates inMCF-7 tumor bearing CF-17 SCID mice. Data are obtained from averagingmultiple microPET image slices in the selected organs and tumor, andpresented as mean standard uptake values (SUV) with error barsrepresenting one standard deviation from the mean of of three differentslices.

FIG. 37 shows biodistribution data of ⁶⁴Cu-DOTA-PNA-K4 conjugates inMCF-7 tumor bearing CF-17 SCID mice post microPET imaging at 24 h p.i.Data are presented as percent injected dose per gram tissue (% ID/g)with error bars representing one standard deviation from the mean of oftwo living animals.

FIG. 38 shows tumor-to-muscle and tumor-to-blood ratios of⁶⁴Cu-DOTA-PNA-K4 conjugates in MCF-7 tumor bearing CF-17 SCID mice at 24h p.i. Data were calculated from biodistribution results post-microPETimaging. Error bars representing one standard deviation from the mean oftwo living animals.

FIG. 39 shows MicroPET coronal (FIG. 39(A)) and transaxial (FIG. 39(B))images of two mice administered with ⁶⁴Cu-DOTA-SCK(PTD)-PNA50 and⁶⁴Cu-DOTA-SCK(PTD)-PNA50S (left and right mouse, respectively. Ca. 150μCi/100 μl injected per mouse) at 1 h p.i. The intensity of the imageswas scaled by max/min of frame. Tumors are indicated by solid whitearrows.

FIG. 40 shows tumor-to-muscle and tumor-to-blood ratios ofPNA-conjugated SCKs in comparison with the free PNAs. Data werecalculated from post microPET imaging biodistribution results from twoliving animals.

FIG. 41 shows post microPET imaging biodistribution data obtained frommice administered with ⁶⁴Cu-DOTA-SCK(PTD)-PNA50, ⁶⁴Cu-DOTA-PNA50, and⁶⁴Cu-TETA-SCK. Data are presented as percent injected dose per organ (%ID/organ).

DETAILED DESCRIPTION OF THE DISCOVERY

This discovery provides new beneficial therapeutic nanoparticleconjugates and their use as molecular probes for the early stagedetection of cancer and/or as therapeutic agents for the treatment ofcancer. More in particular this discovery provides a unique targetingligand optionally associated with biomedical nanoparticles which isuseful in a medical diagnostic method to diagnose cancer or in atherapeutic method to treat cancer in a living mammal such as cancer ina living human.

This discovery provides an enhanced functional delivery system andprocess for effectively delivering particle conjugates into cells aseffective cell, gene therapy, drug therapy and radiotherapy to livingmammals such as to living humans.

The inventors discovered cell permeable SCK-PNA nanoconjugates having asan intracellular targeting ligand a PNA or another nuclease resistantoligonucleotide analog that does not activate the degradation of themRNA by RNase H, such as MOE-mRNA or LNA, having a functional unr mRNAbinding sequence such as PNA50 (see SEQ ID 3) or a sequence that bindsselectively to a unique or overexpressed marker mRNA specific to thecancer or disease state. The inventors have shown utility here in thattheir nanoconjugates with PNA50 concentrate in MCF-7 cells in vitro andin a mouse xenograft. The uniquely or overexpressed mRNA is referred toherein as a recognition element.

SCK nanoparticles are especially useful in this discovery in that SCKnanoparticles provide high valency and functionality for highlyfunctionally capable association with chelators and chemotherapeuticdrugs, cytotoxic drugs and prodrugs. The increased valency is highlydesired in that an increased number of functionalities can beadvantageously capably attached to or associated with the nanoparticlesthereby providing increased efficacy to the conjugated nanoparticles.

SCK nanoparticles useful herein include, but are not limited to,self-assemblying micellar assemblies of amphiphilic copolymers that havebeen stabilized through the incorporation of covalent cross-links in theshell layer. More particularly the particles comprise amphiphiliccopolymers, having a cross linked shell domain and an interior coredomain. Also provided are compositions comprising such particlesincluding pharmaceutical compositions, methods of making the presentparticles and methods of using such particles for example for effectivedelivery of pharmaceutically active agents. In an aspect a usefulparticle includes a particle comprising an amphiphilic copolymer andhaving a core and a crosslinked shell which differs from the core inhydrophilicity and hydrophobicity, the shell comprising a region of thecopolymer which differs in hydrophilicity and hydrophobicity fromanother region of the copolymer in the core, the copolymer being crosslinked in the region within the shell, the copolymer region in the shellhaving a degree of crosslinking ranging from about 1% to about 80%, thecopolymer being selected from among amphiphilic copolymers physicallyconducive to forming micelles prior to crosslinking. In an aspect anillustrative useful multifunctional nanodevice platform comprises theplatforms disclosed in U.S. Pat. No. 6,471,968 which issued to Baker,Jr., et al on Oct. 29, 2002 and particles disclosed in U.S. Pat. No.6,383,500 which issued to Karen L. Wooley et al On May 7, 2002. U.S.Pat. No. 6,471,968 and U.S. Pat. No. 6,383,500 are incorporated hereinin their entirety by reference.

As used herein the term “SCK” means shell crosslinked knedel shellcrosslinked nanoparticle.

As used herein, the term “nanodevice” refers to and includes, but is notlimited to, small (e.g., invisible to the unaided human eye)compositions containing or associated with one or more useful “agents.”In its simplest form, the nanodevice comprises a physical compositionsuch as a nanoparticle or a dendrimer associated with at least one agentthat provides biological functionality (e.g., a radionuclide or atherapeutic agent).

In an aspect, a useful permeation peptide is the transduction domain ofthe HIV-1 TAT protein which is used to capably enable cell membranetransduction. (Seq. Id. 1)

As used herein the term “PTD” means Protein Transduction Domain andindicates the HIV-1 TAT protein transduction domain (Seq. Id. No. 1)

In another aspect, a useful permeation peptide is the nuclear localizingsequence, herein referred to as “NLS” (Seq. KPKKKRKV), or the Lys4tetrapeptide, which are used to capably enable cell membranetransduction of attached molecules or nanodevices.

Without being bound by theory these permeation peptide(s) are believedto capably and effectively enable reversible intracellularmobility/translocation of attached molecules and naodevices andadvantageously thereby allow useful cell targeting of molecules ornanodevices through binding to intracellular targets such as mRNA orother cell receptors, such as intracellular cytostolic proteins ormembrane receptors, thereby increasing the numbers of cancer ordisease-specific receptors.

In an aspect, the nanodevice is targeted to that target mRNA which isuniquely expressed or is significantly overexpressed in a living mammalafflicted with a disease such that the unique expression orsignificantly overexpression is significantly indicative of the presenceof a disease or disease state in that living mammal having that uniqueor over expression of mRNA. Without being bound by theory, this enhancedtargeting availability is believed to provide an enhanced method andsystem for the diagnosis and detection of cancer. Such targeting of adiseased cell is effected by an antisense PNA or other nucleic acidanalog on the nanoconjugate that is complementary to a unique oroverexpressed mRNA sequence that is unique to the diseased cancer cellin the living mammal. By complementary it is meant that the PNA oranalog has a sequence that can recognize and bind to the mRNA which isassociated with a disease by Watson Crick base pairing. Alternativelythe targeting is effected by the PNA or other nucleic acid analog actingin a stand alone capacity. In an aspect the living mammal is a livinghuman.

As used herein, the phrase “ . . . does not activate the degradation ofthe mRNA by RNase H . . . ” is meant that the moiety “avoids mRNAdegradation by RNase H” and the mRNA remains significantly viable andfunctional.

In some aspects of the present discovery, the living human is suspectedof having cancer and a diagnosis is desired to confirm that, or in someaspects the presence of cancer is confirmed and a therapeutic treatmentis desired.

In some aspects the cancer includes, but is not limited to, lung,breast, melanoma, colon, renal, testicular, ovarian, lung, prostate,hepatic, germ cancer, epithelial, prostate, head and neck, pancreaticcancer, glioblastoma, astrocytoma, oligodendroglioma, ependymomas,neurofibrosarcoma, meningioma, liver, spleen, lymph node, smallintestine, colon, stomach, thyroid, endometrium, prostate, skin,esophagus, and bone marrow cancer. In some embodiments, compositionscomprising nanodevices, and any other desired components (e.g.,pharmaceutically acceptable carriers, adjuvants and excipients) areadministered to the subject. The present discovery is not limited by theroute of administration. Such efficacious administration routes include,but are not limited to, endoscopic, intratracheal, intralesion,percutaneous, intravenous, subcutaneous, and intratumoraladministration.

Briefly, as is described herein, the inventors synthesized PNAs by usingautomated Fmoc solid phase synthesis on an ABI 8909 synthesizer usingcommercially available PNA and amino acid monomers andtri-tert-butyl-DOTA. PNA50 (see SEQ ID 3) is a sequence that iscomplementary to (i.e., antisense), and shows high affinity (K_(D) 20pM) for site 2828-2845 of the unr mRNA (GI: 20070240) that isoverexpressed in MCF-7 breast cancer cell line, as discovered by theinventors by a reverse transcriptase random oligodeoxynucleotide libraryassay (RT-ROL).

PNA50S (see SEQ ID 4) is the sense sequence (has the same sequence assite 2828-2845) and is not complementary and shows little affinity(K_(D)>10,000 pM) for the unr mRNA. The mRNA-recognition element is notlimited to PNA, but in an aspect is a nuclease resistant oligonucleotideanalog such as MOE-mRNA or LNA. In an aspect the mRNA-recognitionelement is MOE-mRNA. In an aspect the mRNA recognition element is LNA.

Bifunctional chelators (“BFC”) used to prepare new bioconjugates labeledwith radionuclides of 2+ and 3+metals include functional derivatives of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, CAS Reg# 60239-18-1), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (TETA, CAS Reg # 339091-75-7), diethylenetriaminepentaacetic acid(DTPA, CAS Reg # 67-43-6), wherein CAS means Chemical Abstracts Service.

The aforementioned three BFCs (herein after respectively DOTA, TETA, andDTPA) are commercially available from Macrocyclics Inc. (2110 ResearchRow, Suite 425, Dallas Tex. U.S.A.), Sigma-Aldrich Chemical Co. (3050Spruce St., St. Louis Mo. U.S.A.), and/or Strem Chemicals, Inc. (7Mulliken Way Newburyport Mass. U.S.A.). If desired, one of skill in theart can synthesize BFCs useful herein after reading this application.

In an aspect functional group(s) are effectively used for conjugation ofradionuclides to SCK nanoparticles, including but not limited tocarboxylic acid or amino groups. They are conjugated through multi-atomspacers (for example —(CH₂)_(n)—) to the nitrogen or carbon atoms ofBFCs wherein n is an integer independently varying from 2 to about 10.In a further aspect, functional groups are useful for effectiveconjugation of a pharmaceutical agent such as a cytotoxic prodrug andradiopharmaceutical to the particle conjugate.

As to radionuclides, useful but not limiting radionuclides include 2+and 3+ metal radioisotopes of biomedical interests (transition metals,lanthanides and actinides). The radionuclides may further extend tousing radioiodine upon successful introduction of tyrosine orp-hydroxystyrene into the SCK.

The inventors provide data demonstrating the tumor-targeting(diagnostic) capability of a ⁶⁴Cu-labeled SCK(PTD)-PNA nanoconjugate inMCF-7 tumor-bearing laboratory living mice as evidence of the credible,specific and general utility of this discovery.

The inventors synthesized and recovered (DOTA-SCK(PTD)-PNA50) and(DOTA-SCK(PTD)-PNA50S) as shown hereinafter. These nanoconjugates wereeach respectively recovered and purified as the titled respectivematerial.

Nanoconjugate (DOTA-SCK(PTD)-PNA50) has a targeting sequence (PNA50)that is complementary (antisense) to the unr mRNA, which the inventorsdiscovered is overexpressed in living MCF-7 breast cancer cells.

Nanoconjugate (DOTA-SCK(PTD)-PNA50S) has a control sequence (PNA50S)that is not complementary (sense). Both these nanoconjugates wererespectively radiolabeled by the inventors with ⁶⁴Cu, and were evaluatedin a breast cancer mouse model (MCF-7 xenograft).

The inventors' data from that evaluation (of the aforementionednanoconjugates) show that the MCF-7 tumor specificity of bothnanoconjugates is about the same as the specificity of free respectiveDOTA-PNA50-K4 and DOTA-PNA50S-K4. Furthermore, compared to free PNAs andfree SCKs, the SCK nanoconjugates surprisingly showed more optimalfeatures (e.g., lower accumulation in the mice liver and kidneys) foreither imaging by PET or therapy.

Advantageously the transduceable medical system of this discovery iscapable of reversibly transducing peptides, proteins and syntheticconstructs, which are not normally able to access intracellularcomponents, across the cellular plasma membrane of living cells. Thisutility provides an enhanced technique available as a research tool.

As an aspect of the present discovery the inventors identified andselected the MCF-7 breast cancer cell line as a target cell line becausethe inventors discovered by analysis of SAGE libraries that that thisbreast cancer cell line possesses an overexpressed (10-fold) mRNA (unr)that is also very abundant (about 5,000 copies per cell). This inventionis not limited to MCF-7 cells and unr mRNA as over-expressed or uniquelyexpressed mRNAs can be found in other cancer cells by analysis of SAGElibraries or by DNA chip analysis.

The inventors identified sequences in the folded unr mRNA that aretightly bound by a complementary PNA (in this instance PNA50) by aRT-ROL assay. These are targeted sequences.

The inventors confirmed tight binding of PNA50 (approx. 10 pM K_(D)) bya newly developed binding assay (Dynabead assay). The inventorsconfirmed that the sense sequence that is complementary to PNA50(PNA50S) does not bind significantly.

The inventors confirmed binding to MCF-7 cells in cell culture bypreparing and using fluorescently tagged PNA50 and PNA50S with attachedNLS peptide sequence (F-PNA50x-NLS). X is either 50 or 50S.

The inventors confirmed selective targeting of MCF-7 tumor cells in miceby a ⁶⁴Cu-tagged DOTA-PNA50x-K4, where DOTA binds the ⁶⁴Cu, and K4 isfour lysines for cell permeation. A selectivity of about 3:1 wasobserved for PNA50 as compared to PNA50S.

The inventors synthesized PNA50x-Lys for attachment to ⁶⁴Cu-SCK-PTD andgenerated MCF-7 tumor detection device, which showed a selectivity levelsimilar to that of free PNA. X is either 50 or 50S.

As used herein the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. As used herein,the term “unr mRNA” means mRNA deriving from the transcription of theunr gene, the transcription unit located immediately upstream of theN-ras gene in the genome of several mammalian species.

As used herein the therm “MOE-mRNA” means 2′-methoxyethyl mRNA which isa class of nucleic acid analogs complementary to the RNA template regionand substituted in 2′ position with a 2-methoxyethyl group.

As used herein the term “ODN” means oligodeoxynucleotide.

As used herein the term “cDNA” means complementary DNA which is DNA thatis synthesized, by reverse transcriptase, from a mRNA template.

As used herein, the term “PNA” means Peptide Nucleic Acid which is asynthetic DNA mimic featuring a chain with repeating N-(2-aminoethyl)glycine units instead of the sugar-phosphate backbone. Commercialmonomers used to synthesize PNAs areN-aminoethyl-N-(adenin-9-ylacetyl)glycine, herein referred to as “A” inPNA sequences, N-aminoethyl-N-(cytosin-1-ylacetyl)glycine, hereinreferred to as “C” in PNA sequences,N-aminoethyl-N-(guanin-9-ylacetyl)glycine, herein referred to as “G” inPNA sequences, and N-aminoethyl-N-(thymin-1-ylacetyl)glycine, hereinreferred to as “T” in PNA sequences.

Without being bound by theory, it is believed that PNA mimics thebehavior of DNA and binds complementary nucleic acid strands and becauseof the neutral backbone of PNA results in stronger binding and greaterspecificity than normally achieved.

As used herein the term “PNA-K4” means a PNA-conjugate with four lysinesat the C terminus.

As used herein the term “LNA” means Locked Nucleic Acid which is an RNAderivative in which the ribose ring is constrained by a methylenelinkage between the 2′-oxygen and the 4′-carbon. LNA monomers arebicyclic compounds structurally similar to RNA nucleosides. In LNA thefuranose ring conformation is restricted by a methylene linker thatconnects the 2′-O position to the 4′-C position. Thus nucleic acidscontaining one or more LNA modifications are referred to herein as LNA.

As used therein the term “RT-ROL assay” means Reverse TranscriptaseRandom Oligodeoxynucleotide Library assay. In this assay antisensebinding sites are identified by the ability of a random 9-meroligonucleotide library terminating a specific base and a PCR tag toprime complementary DNA synthesis by reverse transcriptase. The cDNA isthen amplified by PCR with a radiolabeled primer having the same senseas the RNA, and an unlabelled primer having the same sequence as the PCRtag.

As used herein the term “PCR” means Polymerase Chain Reaction which is atechnique to amplify a specific region of single or double-stranded DNA.

As used herein the term “RT-PCR” means Reverse Transcriptase PolymeraseChain Reaction (aka RNA phenotyping, RNA-PCR or message amplificationphenotyping) which is a method for amplification of a specific mRNA byprior use of reverse transcriptase to form a cDNA, then use of PCR toamplify the cDNA.

As used herein the term “SAABS” means Serial Analysis of AntisenseBinding Sites.

As used herein the terms “PET” and “SPECT” mean Positron EmissionTomography and Single Photon Emission Computed Tomography, respectively.

As used herein, microPET (microPET®, Concorde Microsystems Inc.,Knoxville, Tenn. USA) is a non limiting example of small animal imagingdevices with high resolution useful in detecting the invention in vivo.Other manufacturer also offers other small animal scanner, for exampleMosaic® from Phillips.

The term “biologically active,” as used herein, refers to a protein orother biologically active molecules (e.g., catalytic RNA) havingstructural, regulatory, or biochemical functions of a naturallyoccurring molecule.

The term “shell domain” means the outermost domain or peripheral layerof a particle of the present discovery. When produced in a hydrophiliccontinuous medium, the peripheral layer of the micelles giving rise tosuch particles (and the peripheral layer of the particles themselves) issubstantially hydrophilic; when produced in a hydrophobic continuousmedium, the peripheral layer of the micelles giving rise to suchparticles (and the peripheral layer of the particles themselves) issubstantially hydrophobic.

The term “interior core domain” means the domain of a micelle orparticle interior to the shell domain.

The term “amphiphilic copolymer” means a copolymer which contains atleast one hydrophilic domain and at least one hydrophobic domain.

The term “block copolymer” means a linear polymer having regions orblocks along its backbone chain which are characterized by similarhydrophilicity, hydrophobicity, or chemistry. The term “diblockcopolymer” means a block copolymer comprising two blocks. The term“triblock copolymer” means a block copolymer comprising three blocks.The term “multiblock copolymer” means a block copolymer comprising aplurality of blocks.

The term “aggregation number” means the average number of amphiphiliccopolymer molecules per micelle or particle.

The term “glass transition temperature”, herein referred to also withthe symbol “T_(g)”, means the temperature at which a polymer changesfrom a glassy, hard state to a flexible state.

The term “micelle” includes without limitation micelles having shapes ofspheres, cylinders, discs, needles, cones, vesicles, globules, rods,elipsoids, and any other shape that a micelle can assume under theconditions described herein, or any other shape that can be adoptedthrough aggregation of the amphiphilic copolymers.

The term “particle” means a particle and the largest domain of which isless than one micron includes, but is not limited to, nanoparticles.Dendrimer may be employed as particles herein. Their branching shapeprovides them with large surface area to which to attach therapeuticagents or other biologically active molecules. In an aspect onedendrimer is associated or carries a molecule that recognizes cancercells, a therapeutic agent to kill those cells, and a molecule thatrecognizes the signals of cell death. The shape of the particlesincludes spheres, cylinders, discs, needles, cones, vesicles, globules,rods, elipsoids, and any other shape that a micelle can assume under theconditions described herein, or any other shape that can be adoptedthrough aggregation of the amphiphilic copolymers.

The term “monomer” means a molecule which is capable of combining with anumber of like or unlike molecules to form a polymer.

The term “isolated” with respect to a composition such as a protein thatthe material has been prepared and recovered in a state substantiallyfree of any and all components which might be normally present with itin nature.

The term “pharmaceutically active agent” means any physiologically orpharmacologically active substance that produces a local or systemiceffect in animals, including warm-blooded mammals, humans, and primates;avians; household, sport, and farm animals; laboratory animals; fishes;reptiles; and zoo animals. As used herein, the term “test compound” or“candidate” refers to any chemical entity, pharmaceutical, drug and thelike that can possibly be used to treat or prevent a disease, illness,sickness or disorder of bodily function. Test compounds include bothknown and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by screening using the screening methodsand screening compositions of this discovery. A known therapeuticcompound refers to a therapeutic compound that has been shown to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense andincludes environmental and biological samples. Biological samplesinclude viable and representative samples including animal, includinghuman, fluid (e.g., blood, plasma and serum), solid (e.g., stool),tissue, liquid foods (e.g., milk) and solid foods (e.g., vegetables).

The term “permeable” pertains to the property of a domain wherebyselected atoms or molecules can pass through the domain.

As used herein, the term “peptide” is any of a group of compoundscomprising two or more amino acids linked by chemical bonding betweentheir respective carboxyl and amino groups. The term “peptide” includespeptides and proteins that are of sufficient length and composition toaffect a biological response, e.g. antibody production or cytokineactivity whether or not the peptide is a hapten. The term “peptide”includes modified amino acids, such modifications including, but notlimited to, phosphorylation, glycosylation, pegylation, lipidization andmethylation.

As used herein, the term “polypeptide” is any of a group of natural orsynthetic polymers made up of amino acids chemically linked togethersuch as peptides linked together. The term “polypeptide” includespeptides, proteins, translated nucleic acid and fragments thereof.

As used herein, the term “polynucleotide” includes nucleotide sequencesand partial sequences, DNA, cDNA, RNA variant isoforms, splice variants,allelic variants and fragments thereof.

The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein when referring to a translated nucleic acid (e.g.a gene product).

As used herein, the term “nucleic acid” refers to oligonucleotides orpolynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid(RNA) as well as analogs of either RNA or DNA, for example made fromnucleotide analogs any of which are in single or double stranded form.As used herein the phrase “The Expedite 8909 Nucleic Acid SynthesisSystem” includes the 8909 synthesis system as well as synthesis systemswhich effectively synthesize DNA and RNA oligonucleotides as well aspeptide nucleic acids (PNA) The 8909 system synthesizes short primersand probes as well as sequences exceeding 100-mer in scales ranging from50 nmol to 15 μmol. This is available from SCIENTIFIC INC., Suite 111,2131 Pleasant, Hill Rd Duluth Ga. 30096.

As used herein the term “nuclease resistant oligonucleotides” includesoligonucleotides usefully modified to exhibit resistance to nucleasesand to hybridize with appropriate strength and fidelity to its targetedmRNA. Useful nuclease resistant oligonucleotides include those havingvarious 2′-substitutions have been introduced in the oligonucleotides.The nuclease resistance of these compounds has been increased by theintroduction of 2′-substituents such as halo, alkoxy and allyloxygroups.

As used herein the term “oligonucleotide” refers to a plurality ofnucleotides joined together in a specific sequence from naturally andnon-naturally occurring nucleobases. Useful nucleobases inside thesejoined through a sugar moiety via phosphorus linkages, and includeadenine, guanine, adenine, cytosine, uracil, thymine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyladenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosineand 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine,8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyladenine and other 8-substituted adenines, 8-halo guanines, 8-aminoguanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine andother 8-substituted guanines, other aza and deaza uracils, other aza anddeaza thymidines, other aza and deaza cytosines, other aza and deazaadenines, other aza and deaza guanines, 5-trifluoromethyl uracil and5-trifluoro cytosine. The sugar moiety may be deoxyribose or ribose.Useful oligonucleotides comprise modified nucleobases or nucleobaseshaving other modifications, and in particular modifications thatincrease their nuclease resistance.

As used herein, the terms “bifunctional chelator” or “BFC” refer toorganic compounds containing two or more donor atoms spatially situatedso as to form coordinate bonds with the same metal atom (first function)and a chemical group suitable for conjugation to a biologically activemoiety (second function). Useful chelators include those which are“multidentate”, which means they have multiple donor atoms available forsimultaneous complexing with a suitable metal atom.

As used herein, the term “patient” and “subject” are synonymous and areused interchangeably herein and refer to living organisms.

As used herein, the term “expression” includes the biosynthesis of aproduct as an expression product from a gene such as the transcriptionof a structural gene into mRNA and the translation of mRNA into at leastone peptide or at least one polypeptide.

As used here, the terms “isoforms” and “splice variant” includesalternative occurring forms of RNA transcribed from a genome as well aspolypeptides encoded by a splice variant of mRNA transcribed from agene.

As used herein, the term “therapeutic agent” is any molecule or atomwhich is conjugated, fused or otherwise affixed to a tumor targetingmoiety to produce a conjugate which is useful for cancer therapy.

As used herein, the term “label” includes a detectable label whichincludes any radiolabeled species including ⁶⁴Cu, fluorescent agentssuch as fluorescent proteins, and paramagnetic ions.

As used herein, the term “mammal” includes living mammals includingliving humans and living non-human animals such as murine, porcine,canine, rodentia and feline.

As used herein, the term “antisense” means a polynucleotide or analogwhose sequence of bases is complementary to messenger RNA.

As used herein, the term “sense” means a polynucleotide or analog whosesequence of bases is identical to messenger RNA.

As used herein a “therapeutic amount” is an amount of drug ordrug-conjugated moiety which produces a desired or detectabletherapeutic effect on or in a mammal.

As used herein, the term “probe” includes DNA or RNA molecules ofspecific base sequence, labeled either radioactively or with afluorophore, that are used to detect the complementary base sequence byhybridization.

As used herein pZero plasmid is used for a SAGE analysis fromInvitrogen.

As used herein, the term “radiolabeled counterpart(s)” includesrespective radiolabeled compounds.

As used herein, the term “detectably labeled” includes the respectiveradiolabeled compounds having an effective amount of an emittingradiolabel therewith and suitably accepting an emitting radiolabel foruse in effective administration to living mammals.

As used herein, the symbol “Cu” means copper, “C” means carbon, “H”means hydrogen, “0” means oxygen, “N” means nitrogen, “Br” meansbromine, “D” means deuterium, “Cl” means chlorine, “P” meansphosphorous, “I” means iodine, “Na” means sodium, “Ar” means argon, “Kr”means krypton, “He” means helium, “Ne” means neon, “Ni” means nickel,“Li” means lithium.

As used herein in peptide sequences the codes “K” and “Lys” mean Lysine,“C” and “Cys” mean Cysteine, “Y” and “Tyr” mean Tyrosine, “P” and “Pro”mean Proline, “R” and “Arg” mean Arginine, “V” and “Val” mean Valine,“Q” and “Gln” mean Glutamine, “G” and “Gly” mean Glycine.

As used herein, the term “administration” includes the effective givingof a compound or moiety by any useful means to a living mammal and itssuccessful introduction into the mammal such as in its gastrointestinaltract or in a blood lumen of the mammal in an effective method whichresults in that compound, its salt, its ions, metabolites or derivativesthereof being made biologically available to that mammal receivingadministration of compound for medicinal use. In an aspect the mammal isa human. In an aspect the mammal is a nonhuman such as a rodent, acanine, or a feline. In an aspect the compound is made biologicallyavailable to the gastro intestinal tract of a living mammal patient.

As used herein, the expression “pharmaceutically acceptable” applies toa composition or its radiolabeled counterpart which contains compositioningredients that are compatible with other ingredients of thecomposition as well as physiologically acceptable to the recipient, e.g.a mammal such as a human, without the resulting production of excessiveundesirable and unacceptable physiological effects or a deleteriousimpact on the mammal being administered the pharmaceutical composition.In an aspect, a composition for use comprises one or more carriers,useful excipients and/or diluents.

As used herein, the term “dosage” includes that amount of compoundwhich, when effectively administered to a living mammal, provides aneffective amount of biologically available compound to the livingmammal.

As used herein the term “patient” includes a living human subject and ahuman individual. In an aspect the patient includes a human, and aliving non-human such as feline, canine, horse and murine.

Particle based moieties useful in the polymer conjugates herein includethose particles disclosed in U.S. Pat. No. 6,383,500 which issued toKaren L. Wooley et al. on May 7, 2002 (hereinafter the '500 patent)which is incorporated herein in its entirety by references. Theparticles of the '500 patent comprise amphiphilic copolymers and have acrosslinked shell domain, which can be permeable, and an interior coredomain. Such particles can comprise a hydrophilic, crosslinked,permeable shell domain and a hydrophobic interior core domain. Theamphiphilic copolymers of the particles can be crosslinked viafunctional groups within the hydrophilic shell domain, for example bycondensation reactions, addition reactions, or chain polymerizationreactions.

In an aspect of the present discovery, the hydrophobic interior coredomain of the particles disclosed in the '500 patent can also becrosslinked via functional groups in their hydrophobic domains.

In an aspect of the present discovery, the particles disclosed in the'500 patent comprising amphiphilic copolymers having a crosslinked shelldomain and an interior core domain comprise a hydrophobic, crosslinkedshell domain, which is permeable, and a hydrophilic interior coredomain. The amphiphilic copolymers of such particles can be crosslinkedvia functional groups within the hydrophobic shell domain, for exampleby condensation reactions, addition reactions, or chain polymerizationreactions. In another embodiment of the present discovery, thehydrophilic interior core domain of such particles can also becrosslinked. In this case, the amphiphilic copolymers can be crosslinkedvia functional groups in their hydrophilic domains.

In an aspect, useful nanoparticles comprise an inner core and an outercoating. The particles are attached to a cancer targeting moiety thatinteract preferably with cancer calls. The particles are also affixedwith a substance akin to a dye that makes them visible on magneticresonance imagining or are labeled with an appropriate emitting ligandsuch as radioactive copper-64.

In yet another aspect, the particles of the present discovery comprisealiphatic copolymers, comprising an outermost crosslinked domain, whichcan be permeable, a series of additional crosslinked (permeable)domains, and a domain interior to each of the crosslinked (permeable)domains, producing an “onion-like” structure.

The inventors have discovered a method for identifying a mRNA forselective targeting and then identifying the PNAs that will bind to it.

In practicing this method, one first identifies the mRNA for targeting.

Basically one uses a SAGE or DNA chip to quantify gene expression in thetarget cell, compares the gene expression profile to all expressiondatabases and identifies a sequence that is most differentiallyexpressed and is in the highest amount or is uniquely expressed toidentify an mRNA of interest,

obtains a clone containing the cDNA for the mRNA of interest andproduces the mRNA in vitro by RNA polymerase,

maps accessible sites by either the modified RT-ROL assay and/or SAABSassay,

screens potential ODNs by the Dynabead dot blot assay,

quantifies the binding of ODNs by the Dynabead direct binding assay with³²P-labeled ODN,

synthesizes and recovers Cys-Tyr-PNA-Lys4 corresponding to the tightestbinding ODNs (or with another permeation peptide in place of Lys4)

quantifies binding of the hybrid PNAs by the Dynabead direct bindingassay with radioiodinated PNA,

conjoins the highest affinity PNAs to fluorescein and DOTA forfluorescence assays of cell binding in vitro or in vivo (mousexenograft) and

conjoins PNAs with the highest affinity to SCK nanoparticles through anappended lysine or other suitable accommodating site-specific couplingmoiety.

As used herein the term “SAGE” means Serial Analysis of Gene Expressionwhich is a method to efficiently count large numbers of mRNA transcriptsby sequencing short tags.

Radionuclides:

In an aspect, a diagnostic imaging composition is provided comprising apolymer conjugate, a chelator and a radionuclide. Typically a chelatoris employed to functionally and capably associate a radionuclide withthe polymer conjugate. The radionuclide may be conjugated with thepolymer or with the PNA alone.

For example, a radioisotope can be appended to the polymer conjugateusing techniques known in the art, for example, techniques analogous tothose described in Lewis et al. (Bioconjug Chem 2001, 12:320-324) and inLiu et al. (Bioconjug Chem 1997, 8:621-636).

The polymer conjugate can be conjugated with a chelating group which isthen labeled with a radionuclide, such as a metallic radioisotope. Suchchelating groups are well known in the art and include polycarboxylicacids such as for example ethylenediaminetetraacetic acid (EDTA, CAS Reg# 60-00-4), derivatives of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA, CAS Reg# 60239-18-1), 1,4,8,11-Tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (TETA, CAS Reg # 339091-75-7), diethylenetriaminepentaacetic acid(DTPA, CAS Reg # 67-43-6) and the crossbridged CB-TE2A. and the like, oranalogs or homologs thereof, as well as the chelating groups disclosedin Anderson and Welch (Chem Rev. 99: 2219-2234, 1999) and Jurisson andLydon (Chem. Rev. 99: 2205-2218, 1999).

A chelating group for the radionuclide therein may be attached directlyto the polymer conjugate by means of a divalent or bifunctional organiclinker group. Such bifunctional linker groups are well known in the artand are preferably less than about 50 angstroms in length. Illustrative,nonlimiting examples of useful suitable linker groups include2-carboxymethyl, 3-carboxypropyl, 4-carboxybutyl, and the like.

A bifunctional chelator is of value in this discovery in this aspect. Asused herein, the term “bifunctional chelator” refers to any organiccompound containing two or more donor atoms spatially situated so as toform coordinate bonds with the same metal atom (first functionality) anda functional group suitable for the conjugation to the targeting moiety(second functionality). Useful chelators are those which are“multidentate”, which means they have multiple donor atoms available forsimultaneous complexing with a metal atom. If desired, one or morebifunctional chelators may be employed.

For diagnostic applications herein, acyclic bifunctional chelators maybe satisfactory. For therapy type applications herein macrocyclicbifunctionals are better suited.

In an aspect, a radioactive metal ion, such as ⁶⁴Cu²⁺, is attached to apolymer conjugate via a bifunctional chelator, the latter functioning asa metal coordinating bifunctional chelator. In an aspect the radioactivemetal ion is said to be associated with the polymer conjugate and themetal coordinating bifunctional chelator produces that association.

Without being bound by theory, the purpose of the bifunctional chelator,is to maintain a stable complex with the radioactive metal ion toproduce a high-contrast image and, for radiotherapy, to prevent leakagespread of radioactivity into healthy (non-target) organs and tissuesthroughout the living mammalian body.

The emissions from the radioactive metal allow both 3-D visualization oftumors through PET, SPECT, and microPET, and therapy of tumors due tospecific delivery of a cytotoxic radiation dose to tumor cells.

The reactions between the chelator, the polymer conjugate, and thedetectable radionuclide are carried out using known methods, andpreferably are performed at a pH at which the polymer conjugate isstable and under condition effective to enable the efficient carryingout of the reactions. Illustratively in a method, a complex is formedbetween the chelator and a detectable radionuclide prior to couplingwith the polymer conjugate. In another method, a chelator is complexedfirst with a non-detectable metal ion and then with the polymerconjugate. The non-detectable metal ion may subsequently be replaced bythe desired detectable element via a transmetallation process. It isunderstood that all reactions and synthesis conditions employed hereinare those which suitably accommodate the desired reaction and synthesisand allow it to satisfactorily progress to its desired completion.

With respect to radionuclides, PET (including microPET) and SPECT areuseful non-invasive molecular diagnostic imaging (standard) proceduresthat produce (i.e., capture and optionally record) multiple acquisitionsi.e. images of the body's biological functions and in an aspect are usedto determine the extent of malignant disease. In an aspect, theseimaging procedures show the presence and distribution of a radiolabeleddetectable functionally emitting chemical associated with the polymerconjugate acquired at various selected times. Advantageously theseimaging procedures depict metabolic characteristics of tissues andchanges therein.

A “detectable element” as used herein is defined as any element,preferably a metal ion, which exhibits a property detectable intherapeutic or in vivo diagnostic techniques. For example, a nonlimitingexample of a detectable element is a metal ion that emits detectableradiation or a metal ion that is capable of influencing NMR relaxationproperties and that is capable of forming a conjugate or complex withthe described nanoparticle moiety. Suitable detectable metal ions asused herein include, for example, heavy elements or rare earth ions suchas the paramagnetic ions, Gd³⁺, Fe³⁺, Mn²⁺ and Cr²⁺. By way of example,a nonlimiting example of detectable element is a fluorescent metal ions,such as Eu³⁺, and radionuclides, such as gamma-emitting radionuclides,beta-emitting radionuclides, and positron-emitting radionuclides.

Any radionuclide suitable for imaging or therapy can be employed toprepare a functional polymer conjugate. For example, suitablenonlimiting examples of useful radionuclides include: Actinium-₂₂₅,Astatine-₂₂₅, Bismuth-₂₁₂, Bismuth-₂₁₃, Bromine-₇₅, Bromine-₇₆,Carbon-₁₁, Cerium-₁₄₁, Chromium-₅₁, Copper-₆₀, Copper-₆₁, Copper-₆₂,Copper-64, Copper-₆₇, Dysprosium-₁₆₆, Fluorine-₁₈, Gadolinium-₁₅₂,Gadolinium-₁₅₃, Gold-_(195m), Holmium-₁₆₆, Indium-₁₁₁, Indium-_(110m),Iodine-₁₂₃, Iodine-₁₂₄, Iodine-₁₃₁, Iron-₅₅, Iron-₅₉, Lutetium-₁₇₇,Nitrogen-₁₃, Oxygen-₁₅, Palladium-₁₀₃, Radium-₂₂₄, Rhenium-₁₈₆,Rhenium-₁₈₈, Rubidium-₈₁, Rubidium-₈₂, Rubidium-₈₆, Ruthenium-₁₀₃,Ruthenium-₁₀₆, Samarium-₁₅₃, Scandium-₄₆, Tantalum-₁₇₈,Technetium-_(94m), Technetium-_(99m), Thallium-₂₀₁, Titanium-₄₅,Ytterbium-₁₆₉, Yttrium-₈₆, Yttrium-₉₀, and Zirconium-₈₉.

In an aspect, positron emission tomography (PET) comprises detection oftwo gamma-rays deriving from annihilation of positrons emitted fromradionuclides that decay by positron emission and are located within amammalian patient's body.

A large number of scintillation detectors detect these photon pairs andmeasure the sum of radioactivity along many different paths through thepatient undergoing measurement. Appropriate software associated with theinstrument reconstructs a three-dimensional image of the patient and theconcentrations of radionuclides can be expressed in quantitative unitsof radiotracer concentration per ml of tissue.

In an aspect, single photon emission computed tomography (SPECT)comprises detection of single gamma-rays emitted from radionuclides thatdecay by gamma emission and are located within a mammalian patient'sbody.

SPECT imaging comprises external measurement of the single photonemitted anisotropically by a radioactive compound labeled withgamma-emitting radionuclides. Photons are selected by a collimator.Generally collimators for SPECT imaging are lead and comprise thousandsof various shaped parallel channels through which—and only throughwhich—gamma rays are allowed to pass. Generally such collimators arepositioned over a single crystal of NaI contained in the Gamma camera inan arrangement referred to as an Anger camera. The image or acquisitionfrom the camera is the captured image which is presented to a humanoperator as part of the image. In an aspect multi-acquisition is used.In an aspect a multi-acquisition is carried out over an elapsed timeinterval.

In an aspect a PET and/or a SPECT and/or microPET image is taken of amammal after the satisfactory administration of a radionuclide of to themammal.

In an aspect, images are taken over elapsed time in dynamic fashion toassemble a developing or developed scenario of situations in themammalian patient.

Once a radiolabeled compound(s) is adequately administered to a patient,the emitting radioactivity travels through gastro-intestinal tract orthrough the vascular system of the body and localizes in the appropriateareas of the body (based on targeting) and is detected by PET or SPECTscanners.

Typically an adequate amount of time is allowed to pass for the treatedliving mammal to come to an equilibrium state following satisfactoryadministration of the radioligand to the mammal. Typically the mammal isplaced in a position near the PET or SPECT or microPET instrumentallowing satisfactory operation of the instrument. The PET, microPET,and SPECT instruments are equipped with all necessary operable softwareand operation requirements. They are turned on i.e. energized and madeoperable by supplying 100/220 volts electric power to the respectiveinstruments.

Generally after a living mammal has received its administration of theradiolabeled moiety, the mammal is taken to an examination room thathouses the scanner, which has an opening in the middle. In an aspect themammal is moved into the hole of the machine. The images are displayedon the monitor of a computer, suitably equipped and operably coupled tothe scanner instrument for acquiring.

In an aspect a copper radiolabeled material (compound(s)) isadministered to a living mammal patient. In an aspect the radiolabeledemits a functional externally detectable amount of desired radioactivityin the mammal. In a medical aspect the amount of emitted radioactivityis an amount which imparts a diagnostic or therapeutic benefit to themammalian patient having cancer. In an aspect a therapeutic benefit isthat benefit which is medicinally and therapeutically beneficial to theliving mammalian afflicted with cancer. In an aspect a cytotoxic amountis an effective lethal amount of a therapeutic compound whichbeneficially kills or retards cancer cells.

Useful radiochemical methods are found in the textbook Welch M J,Redvanly C S, Handbook of Radiopharmaceuticals: Radiochemistry andApplications, Chichester: Wiley, 2003.

In an aspect a copper radiolabeled material is effectively administeredto a mammal or to a biological sample thereof or there from and thesample is analyzed and a diagnosis is made or obtained. In an aspect, abiological sample of the mammal comprises a representative sample takenof at least one of blood, vessels, atheroma, liver, and other bodytissues a well as biopsies of body organs such as a liver biopsy or amuscle biopsy of a living mammal. In an aspect, the amount of biologicalsample is that amount or volume which is sufficient to provide for aneffective and discerning analysis.

In an aspect a mammal host is selected from at least one of a livinghuman and non human animal such as canine, feline, equestrian, murineincluding dogs, cats, rabbits, guinea pigs, hamsters, mice, rats,rodents, horses, goats, sheep, pigs and cows. In an aspect the mammalhost is a living patient.

In an aspect, depending on its form, the administered formulation issuitably formulated for ease of facilitation of administration and useby the mammal patient and may contain a binder, disintegrating agent,lubricant, sweetener, a liquid carrier.

In an aspect, a copper radiolabeled compound is administered to a mammalas a pharmacologically acceptable composition. Pharmacologicallyacceptable compositions such as solutions of a labeled compound or itssalts can be prepared in water, optionally mixed with a nontoxicsurfactant.

In an aspect, the amount of time elapsing between imaging procedures isa sufficient time which provides for a useful and meaningful comparisonof acquired images.

Accordingly, the discovery includes a pharmaceutical compositioncomprising a labeled compound as described hereinabove; or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable diluent or carrier.

In an aspect a therapeutic rate titration is performed wherein theliving mammalian afflicted with cancer is administered a series ofdosages and respective effects therefrom or thereafter are determined atrespective dosages and times by methods known to those in thepharmacology art. In this manner a therapeutic dosage curve or titrationis obtained for determining dosage for that mammal patient.

Administration may be performed by any effective local or systemicapplication as appropriate. Administration of compositions may be doneby inhalation, orally, rectally or parenterally, such as byintramuscular, subcutaneous, intraarticular, intracranial, intradermal,intraocular, intraperitoneal, intrathecal and intravenous injection.

In an aspect, an internal radiation cancer therapy useful on livingmammals comprises administering anti-cancer compounds syntheticallylabeled with ⁶⁴Cu to such living mammals. In an aspect, a treatment ofmalignant neoplasms in living mammals (human and nonhuman) comprisesadministering anti-cancer compounds labeled with ⁶⁴Cu.

In an aspect, a method for pharmacologically treating a mammalian tumorin a mammal comprises administering to a mammal having a tumor acomposition including a tumor-inhibiting amount of at least one ⁶⁴Culabeled compound. In an aspect the living mammal is nonhuman.

In an aspect, a method for in vivo detection of cancer cell(s) in livingmammalian tissue samples comprises contacting a mammalian tissue samplewith an in vivo effective diagnostic imaging amount of at least one ⁶⁴Culabeled compound for a time and under conditions sufficient andeffective for binding of labeled compound to the cancer cell(s) anddetecting such binding indicative of an association with the presenceand location of cancer in the contacted cell(s). In an aspect thedetecting is by image acquisition. In an aspect the ⁶⁴Cu labeledcompound is a tracer for cancer. In an aspect, the cell(s) is in apreviously obtained biological sample from a mammal. In an aspect suchbinding is indicative of the presence of and location of a cancer cell.In an aspect, the mammal is a living human and the radionuclide is ⁶⁰Cu,⁶¹Cu or ⁶⁴Cu. In an aspect the living mammal is a nonhuman mammal. In anaspect the extent of binding is determined by comparing the amount ofradioactivity administered to the animal with the amount ofradioactivity and location of radioactivity detected by imageacquisition.

In an aspect, a method for determining progression or regression of acancer in a living mammal comprises administering to a living mammal adiagnostic imaging detectable amount of at least one highly purified⁶⁴Cu labeled compound at a first selected time, detecting an image of acancer tissue in the mammal being treated at a second selected (later)time respectively detecting an image of a cancer tissue at both times,comparing the images and determining if the detected cancer tissue inthe image at the later time is bigger or smaller than the detectedcancer tissue in the image at the first time. In an aspect the elapsedtime between the first time and second time is selected to be a timeduration significant amount. In an aspect the living mammal is nonhuman.In an aspect the comparison is used to determine progression orregression of a cancer in a mammal.

Pharmaceutical Compositions

In an aspect a pharmaceutical composition comprises a particle conjugateand a pharmaceutical agent or a PNA and a pharmaceutical agent. In anaspect the pharmaceutical agent comprises at least one of a cancer drug,a prodrug or a radionuclide.

The particle conjugate further comprises a nano-scale particle basedmoiety having associated therewith a permeation peptide (e.g., the HIV-1TAT protein transduction domain) and a nucleic acid analog capable ofbinding to a complementary disease specific mRNA sequence, wherein useof the nucleic acid analog does not lead to the destruction of thedisease specific mRNA sequence, wherein particles comprise amphiphiliccopolymers having a crosslinked shell domain, which can be permeable,and an interior core domain, or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier, excipient, ordiluent.

The pharmaceutically active agent can be contained within the particleor conjugated to it. The pharmaceutically active agent can be present inthe particle dissolved in the crosslinked shell domain (which can bepermeable), or covalently attached to a component of the crosslinkedshell domain, or in the form of a fine dispersion within the crosslinkedshell domain, or on the surface of the crosslinked shell domain.

Alternatively, the pharmaceutically active agent can be present in theparticle dissolved in the interior core domain, or covalently attachedto a component of the interior core domain, in the form of a finedispersion within the interior core domain, or on the surface of theinterior core domain, or at the interface between the crosslinked shelldomain and the interior core domain.

The pharmaceutically active agent can also be present both in thecrosslinked shell domain and in the interior core domain, or covalentlyattached to components of each domain, or in the form of a finedispersion within each domain, or on the surface of each domain.

The pharmaceutically active agent can be introduced to the polymerconjugate in a variety of different ways. For example, in the process offorming particles of the present discovery, the pharmaceutically activeagent can be present in the solvent system employed to form the micellesthat are the precursors to the particles of the discovery. Uponformation of the particles, the pharmaceutically active agent isentrapped therein. Alternatively, pre-formed particles can be suspendedin a solvent containing the active agent, and thus take up thepharmaceutically active agent from solution. In addition, thepharmaceutically active agent can be sprayed in the form of a solutionor a melt onto the surface of the pre-formed particles. In anotherexample, the pre-formed particles can be treated with a vapor containingthe pharmaceutically active agent. The pharmaceutically active agent canalso be vacuum infiltrated into the pre-formed particles.

The pharmaceutically active agent can be associated with or affixed tothe amphiphilic copolymers which comprise the particles of thisdiscovery either chemically or physically. The association or affixingcan be performed either prior to the preparation of the particles orafter the preparation of the particles.

When present in polymer conjugate of the present discovery as describedabove, the pharmaceutically active agent can be released there from. Itis fully expected that such release can be sustained, i.e. notimmediate, but rather over an extended period of time, thereby makingparticles of the present discovery containing pharmaceutically (or otheractive) agents useful as sustained release delivery vehicles.

Pharmaceutically Active Agents

Nonlimiting examples of useful pharmaceutically active agents that canbe used with these polymer conjugates include inorganic and organiccompounds without limitation, including drugs that act on the peripheralnerves, adrenergic receptors, cholinergic receptors, nervous system,skeletal muscles, cardiovascular system, smooth muscles, bloodcirculatory system, synaptic sites, neuroeffector junctional sites,endocrine system, hormone systems, immunological system, reproductivesystem, skeletal system, alimentary and excretory systems, inhibitory ofautocoids and histamine systems. The active drugs that can be deliveredfor the purpose of acting on these recipients include anticonvulsants,analgesics, anti-inflammatories, calcium antagonists, anesthetics,antimicrobials, antimalarials, antiparasitics, antihypertensives,antihistamines, antipyretics, alpha-andrenergic agonist, alpha-blockers,anti-tumor compounds, biocides, bactericides, bronchial dilators,beta-andrenergic blocking drugs, contraceptives, cardiovascular drugs,calcium channel inhibitors, depressants, diagnostics, diuretics,electrolytes, hypnotics, hormonals, hyperglycemics, muscle contractants,muscle relaxants, opthalmics, psychic energizers, parasympathomimetics,sedatives, sympathomimetics, tranquilizers, urinary tract drugs, vaginaldrugs, vitamins, nonsteroidal anti-inflammatory drugs, angiotensinconverting enzymes, polypeptide drugs, and the like.

Nonlimiting examples of useful pharmaceutically active agents that arehighly soluble in water and that can be used in conjunction with theparticles of the present discovery include prochlorperazine edisylate,ferrous sulfate, aminocaproic acid, potassium chloride, mecamylaminehydrochloride, procainamide hydrochloride, amphetamine sulfate,benzphetamine hydrochloride, isoproterenol sulfate, methamphetaminehydrochloride, phenmetrazine hydrochloride, bethanechol chloride,methacholine chloride, pilocarpine hydrochloride, atropine sulfate,scopolamine bromide, isopropamide iodide, tridihexethyl chloride,phenformin hydrochloride, methylphenidate hydrochloride, cimetidinehydrochloride, theophylline cholinate, cephalexin hydrochloride, and thelike.

Exemplary pharmaceutically active agents that are poorly soluble inwater and that can be used in conjunction with the particles of thepresent discovery include diphenidol, meclizine hydrochloride,prochlorperazine maleate, phenoxybenzamine, thiethylperazine maleate,anisindone, diphenadione, erythrityl tetranitrate, digoxin,isoflurophate, acetazolamide, methazolamide, bendroflumethiazide,chlorpropamide, tolazamide, chlormadinone acetate, phenaglycodol,allopurinol, aluminum aspirin, methotrexate, acetyl sulfisoxazole,erythromycin, progestins, sterogenic, progestational, corticosteroids,hydrocortisone hydrocorticosterone acetate, cortisone acetate,triamcinolone, methyltestosterone, 17 beta-estradiol, ethinyl estradiol,ethinyl estradiol 3-methyl ether, prednisolone, 17beta-hydroxyprogesterone acetate, 19-nor-progesterone, norgestrel,morethindrone, norethisterone, norethiederone, progesterone,norgesterone, norethynodrel, and the like.

Examples of other pharmaceutically active agents that can be used inconjunction with the particles of the present discovery include aspirin,boron-containing antitumor compounds, indomethacin, naproxen,fenoprofen, sulindac, indoprofen, nitroglycerin, isosorbide dinitrate,propranolol, timolol, atenolol, alprenolol, cimetidine, clonidine,imipramine, levodopa, chloropromazine, methyldopa,dihydroxyphenylalanine, pivaloyloxyethyl ester of alpha-methyl dopahydrochloride, theophylline, calcium gluconate, ketoprofen, ibuprofen,cephalexin, erythromycin, haloperidol, zomepirac, ferrous lactate,vincamine, diazepam, phenoxybenzamine, diltiazem, milrinone, captopril,madol, quanabenz, hydrochlorothiazide, ranitidine, flurbiprofen,fenbufen, fluprofen, tolmetin, alclofenac, mefenamic, flufenamic,difuninal, nimodipine, nitrendipine, nisoldipine, nicardipine,felodipine, lidoflazine, tiapamil, gallopamil, amlodipine, mioflazine,lisinolpril, enalapril, captopril, ramipril, famotidine, nizatidine,sucralfate, etinidine, tertatolol, minoxidil, chlordiazepoxide,chlordiazepoxide hydrochloride, diazepan, amitriptylin hydrochloride,imipramine hydrochloride, imipramine pamoate, enitabas, verapamil,losartan, and the like. Other beneficial pharmaceutically active agentsknown in the art that can be used in conjunction with the particles ofthe present discovery are disclosed in Pharmaceutical Sciences, 14thEd., edited by Remington, (1979) published by Mack Publishing Co.,Easton Pa.; The Drug, The Nurse, The Patient, Including Current DrugHandbook, by Falconer, et al., (1974-1976) published by SaundersCompany, Philadelphia, Pa.; Medicinal Chemistry, 3rd Ed., Vol. 1 and 2,by Burger, published by Wiley-Interscience, New York; Goodman & Gilman'sThe Pharmacological Basis of Therapeutics, 9th Ed., edited by Hardman,et al., (1996) published by McGraw-Hill, New York, N.Y.; and inPhysicians' Desk Reference, 51st Ed., (1997) published by MedicalEconomics Co., Montvale, N.J.

Prodrugs

Prodrugs are useful in pharmaceutical compositions of this discovery andin an aspect such drugs may be conjugated to the polymer conjugate.Prodrugs are pharmacologically inactive derivatives of active drugs.Prodrugs are designed to maximize the amount of active drug that reachesits respective effective site of action through manipulation of thephysicochemical, biopharmaceutical or pharmacokinetic properties of thedrug. Prodrugs are capably converted into the pharmaceutically activedrug within the body through enzymatic or non-enzymatic reactions afterthe prodrugs are administered to the patient.

In an aspect, a useful drug has cytotoxicity greater than that of theprodrug. Typically, the prodrug has an enzyme cleavable covalent linkbetween a drug and a chemical moiety associated therewith although someuseful moieties of prodrug include the salt form (such as water solubleform) of an active drug molecule. Typically a partly or essentiallywater soluble salt form would be employed, including those moietieswherein there is a covalent link between a drug and chemical moiety andincludes salts of the prodrug such as those which are moderately orhighly water soluble such as alkali metals, ammonium and amine salts andalkaline earth metal salts.

Nonlimiting examples of useful prodrugs include5-(aziridine-1-yl)-2,4-nitrobenzamide,peptidyl-p-phenylenediamine-mustard, benzoic acid mustard glutamates,6-methoxypurine arabinonucleoside, 5-fluorocytosine, glucose,hypoxanithine, methotrexate-alane, N-(94-(-D-galactopyranosyl),benzyloxycarbonyl)-daunorubicine, amygdalin, azobenzene mustards,gamma-glutamyl-p-phenylenediamine mustard, phenolmustard-glucuronide,epirubicin-glucuronide, vinca-cephalosporin,nitrogen-mustard-cephalosporin, phenolmustard phosphate, doxorubicinephosphate, mitomycin phosphate, etoposide phosphate,palytoxin-4-hydroxyphenyl-acetamide, cyclophosphamide isofamide and4-nitrobenzyloxycarbonyl derivatives.

Additional typical useful non-limiting drugs include5-(aziridin-1-yl)-4-hydroxyl-amino-2-nitrobenzamide,phenylenediamine-mustard, ganciclovir triphosphate, adeninearabinonucleoside, triphosphate(araATP), 5-fluorouracid, hydrogenperoxide, superoxide, methotrexate, daunorubicin, cyanide,phenylendiamine mustards, phenyldiamine mustard, phenolmustard,epirubicin, 4-desacetylvinblastine-3-carboxyhydrazide, nitrogenmustards, doxorubicin, mitomycin alcohol, etoposide, palytoxin,melphalan, phosphoamide mustard (+acrolein),5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamine, actinomycin D andmitomycin C.

Cancer Drugs (Cytotoxic)

Drugs cytotoxic to cancer are useful in pharmaceutical compositions ofthis discovery and in an aspect such drugs may be conjugated to thepolymer conjugate or to the PNA.

Typical non-limiting examples of useful cytotoxic drugs includealdesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine,amifostine, anastrozole, arsenic trioxide, BCG Live, bexarotene,bleomycin, calusterone, capecitabine, carboplatin, carmustine,celecoxib, chlorambucil, cisplatin, cladribine, cyclophosphamide,cytarabine, dacarbazine, dactinomycin, darbepoetin alfa, denileukindiftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone, Elliot'sB solution, epirubicin, epoetin alfa, estramustine, etoposide phosphate,exemestane, filgrastim, floxuridine, fludarabine, fulvestrant,gemcitabine, gemtuzumab, goserelin acetate, hydroxyurea, ibritumomabtiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a,interferon alfa-2b, irinotecan, letrozole, leucovorin, levamisole,mechlorethamine, megestrol acetate, melphalan, L-PAM, mercaptopurine6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane,mitoxantrone, androlone phenpropionate, nofetumomab, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase,pegfilgrastim, entostatin, pipobroman, plicamycin, mithramycin, porfimersodium, procarbazine, quinacrine, rasburicase, rituximab, sargramostim,streptozocin, talc, tamoxifen, trastuzumab, tretinoin, uracil mustard,valrubicin, vinblastine, tamoxifen, temozolomide, teniposide,testolactone, 6-thioguanine, thiotepa, topotecan, toremifene,tositumomab, vincristine, vinorelbine and zoledronate.

In another aspect, the present discovery provides a method fordelivering a nucleic acid molecule to a cell, tissue, or organ,comprising contacting the cell, tissue, or organ, in vivo or in vitro,with a composition containing a polymer conjugate of the presentdiscovery and a nucleic acid molecule for a period time sufficient todeliver the nucleic acid molecule to the cell, tissue, or organ. Thenucleic acid molecule can, for example, be present on the surface of theparticle, or within the particle. The nucleic acid molecule can be DNAor RNA for example, an antisense oligonucleotide, a vector, or any othertype of nucleic acid molecule commonly employed in genetic engineeringtechniques

Pharmaceutical Methods

As noted above, polymer conjugates of the present discovery comprising apharmaceutically active agent can be used for sustained release anddelivery of such agents to treat a variety of conditions.

As used herein the term “pharmaceutically active agent” includes atleast one of a prodrug, a cytotoxic drug or a radiopharmaceutical.Optionally if desired one may label prodrug, cytotoxic drug or both.

In one aspect, the present discovery provides a method of effectivelydelivering a pharmaceutical composition chemically or physicallyassociated with the particles of the present discovery. The methodcomprises administering to the mammal a composition comprising theparticles having associated therewith at least one of a permeationpeptide (e.g., HIV-1 TAT protein transduction domain, see SEQ ID 1) anda PNA or another nuclease resistant oligonucleotide analog, such asMOE-mRNA or LNA, having a unr mRNA binding sequence such as PNA50 (seeSEQ ID 3) or any sequence that binds selectively to an unique oroverexpressed mRNA specific to the cancer or disease state.

In another aspect, the present discovery provides a method of deliveringa pharmaceutically active agent to a cell, tissue, or organ, comprisingcontacting the cell, tissue, or organ with an effective amount of apolymer conjugate, the polymer conjugate comprising a particle basedmoiety having associated therewith at least one of a permeation peptide(e.g., the HIV-1 TAT protein transduction domain) and a PNA or anothernuclease resistant oligonucleotide analog, such as MOE-mRNA or LNA,having unr mRNA binding sequence such as PNA50 (see SEQ ID 3) or anysequence that binds selectively to an unique or overexpressed mRNAspecific to the cancer or disease state and a pharmaceutically activeagent, the contact being for a period of time sufficient to introducethe pharmaceutically active agent to the locus of the cell, tissue, ororgan. The method, for example, can comprise contacting the cell,tissue, or organ in vitro or in vivo with the effective amount of theparticles.

As to dosages, formulations, and routes of administration for theprophylaxis or treatment of the conditions referred to above, theparticles of the present discovery can be used as particles per se.Pharmaceutically acceptable salts are particularly suitable for medicalapplications because of their greater aqueous solubility andphysiological compatibility relative to the parent particle. Such saltsmust clearly have pharmaceutically acceptable anions or cations.Suitable pharmaceutically acceptable acid addition salts of theparticles of the present discovery when possible include those derivedfrom inorganic acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric, sulfonic, and sulfuric acids, and organic acidssuch as acetic, benzenesulfonic, benzoic, citric, ethanesulfonic,fumaric, gluconic, glycolic, lactic, lactobionic, maleic, malic,methanesulfonic, succinic, toluenesulfonic, tartaric, andtrifluoroacetic acids. The chloride salt is particularly preferred formedical purposes. Suitable pharmaceutically acceptable base saltsinclude ammonium salts, alkaline metal salts such as sodium andpotassium salts, and alkaline earth salts such as magnesium and calciumsalts.

The polymer conjugates of the present discovery can be effectively andsufficiently administered to a living mammal with an acceptable carrierin the form of a pharmaceutical composition. The carrier must, ofcourse, be acceptable in the sense of being compatible with the otheringredients of the composition and must not be deleterious to therecipient. The carrier can be a solid or a liquid or both, and ispreferably formulated with the particle as a unit-dose composition, forexample a powder or tablet, which can contain from 0.05% to 95% byweight of the active particles. Other pharmacologically activesubstances can also be present, including other particles of the presentdiscovery. The pharmaceutical compositions of the discovery can beprepared by any of the well known techniques of pharmacy, consistingessentially of admixing the components together.

In an aspect, a living patient such as a living mammal such as a livinghuman is treated for cancer according to this discovery. In thatsituation a pharmaceutically useful moiety is administered to thepatient in a “therapeutically effective amount”.

In an aspect pharmaceutical compositions for use herein include inaddition to active ingredient, such as a prodrug, cytotoxic drug orradiopharmaceutical, a pharmaceutically acceptable excipient, carrier,buffer, stabilizer or other materials well known to those skilled in theart. Such materials should be non-toxic and should not interfere withthe efficacy of the active ingredient. The precise nature of the carrieror other material will depend on the route of administration, which maybe oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

These pharmaceutical compositions may be administered by anyconventional means available for administering pharmaceuticalcompositions to living mammals.

The amount of pharmaceutical composition required to achieve the desiredbiological effect will, of course, depend on a number of factors such asthe specific particle or agent chosen, the use for which it is intended,the mode of administration, and the clinical condition of the recipient.

In general, an effective daily pharmaceutical composition dose can be inthe range of from about 5 to about 5,000 mg/kg of bodyweight/day,preferably from about 10 to about 2,000 mg/kg bodyweight/day, morepreferably from about 20 to about 1,000 mg/kg bodyweight/day. This totaldaily dose can be administered to the patient in a single dose, or inproportionate multiple subdoses. Subdoses can be administered 2 to 6times per day. If desired, doses can be in sustained release formeffective to obtain the desired results.

Orally administrable unit dose formulations, such as liquids, tablets,or capsules, can contain, for example, from about 1 to about 5,000 mg ofthe particles, preferably about 2 to about 2,000 mg of the particles,more preferably from about 10 to about 1,000 mg of the particles. In thecase of pharmaceutically acceptable salts, the weights indicated aboverefer to the weight of the particle ion derived from the salt.

Oral delivery of particles of the present discovery can includeformulations, as are well known in the art, to provide prolonged orsustained delivery of the particles to the gastrointestinal tract by anynumber of mechanisms. These include, but are not limited to, pHsensitive release from the dosage form based on the changing pH of thesmall intestine, slow erosion of a tablet or capsule, retention in thestomach based on the physical properties of the formulation, bioadhesionof the dosage form to the mucosal lining of the intestinal tract, orenzymatic release of the particles from the dosage form. The intendedeffect is to extend the time period over which the active particles aredelivered to the site of action (the gastrointestinal tract) bymanipulation of the dosage form. Thus, enteric-coated and enteric-coatedcontrolled release formulations are within the scope of the presentdiscovery. Suitable enteric coatings include cellulose acetatephthalate, polyvinylacetate phthalate, hydroxypropylmethylcellulosephthalate and anionic polymers of methacrylic acid and methacrylic acidmethyl ester.

Pharmaceutical compositions according to the present discovery includethose suitable for oral, rectal, topical, buccal (e.g., sublingual), andparenteral (e.g., subcutaneous, intramuscular, intradermal, orintravenous) administration, although the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular particle which is beingused. In most cases, the preferred route of administration is oral.

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as liquids, capsules, cachets,lozenges, or tablets, each containing a predetermined amount of at leastone type of particle of the present discovery; as a powder or granules;as a solution or a suspension in an aqueous or non-aqueous liquid; or asan oil-in-water or water-in-oil emulsion. As indicated, suchcompositions can be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active particle(s)and the carrier (which can constitute one or more accessoryingredients). In general, the compositions are prepared by uniformly andintimately admixing the active particles with a liquid or finely dividedsolid carrier, or both, and then, if necessary, shaping the product. Forexample, a tablet can be prepared by compressing or molding a powder orgranules containing the particles, optionally with one or more assessoryingredients. Compressed tablets can be prepared by compressing, in asuitable machine, the particles in a free-flowing form, such as a powderor granules optionally mixed with a binder, lubricant, inert diluentand/or surface active/dispersing agent(s). Molded tablets can be made bymolding, in a suitable machine, the powdered particles moistened with aninert liquid diluent.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising particles of the presentdiscovery in a flavored base, usually sucrose, and acacia or tragacanth,and pastilles comprising particles in an inert base such as gelatin andglycerin or sucrose and acacia.

Pharmaceutical compositions suitable for parenteral administrationconveniently comprise sterile aqueous preparations of particles of thepresent discovery. These preparations are preferably administeredintravenously, although administration can also be effected by means ofsubcutaneous, intramuscular, or intradermal injection. Such preparationscan conveniently be prepared by admixing the particles with water andrendering the resulting solution sterile and isotonic with the blood.Injectable compositions according to the discovery will generallycontain from 0.1 to 5% w/w of a particles disclosed herein.

Pharmaceutical compositions suitable for rectal administration arepreferably presented as unit-dose suppositories. These can be preparedby admixing particles of the present discovery with one or moreonventional solid carriers, for example, cocoa butter, and then shapingthe resulting mixture.

Pharmaceutical compositions suitable for topical application to the skinpreferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which can be used include vaseline,lanoline, polyethylene glycols, alcohols, and combinations of two ormore thereof. The active particle is generally present at aconcentration of from 0.1 to 15% w/w of the composition, for example,from 0.5 to 2%.

Transdermal administration is also possible. Pharmaceutical compositionssuitable for transdermal administration can be presented as discretepatches adapted to remain in intimate contact with the epidermis of therecipient for a prolonged period of time. Such patches suitably containparticles of the present discovery in an optionally buffered, aqueoussolution, dissolved and/or dispersed in an adhesive, or dispersed in apolymer. A suitable concentration of the active particle is about 1% to35%, preferably about 3% to 15%. As one particular possibility, theparticle can be delivered from the patch by electrotransport oriontophoresis, for example, as described in Pharmaceutical Research,3(6), 318 (1986).

If desired, the amount of particles that can be combined with carriermaterials to produce a single dosage form to be administered will varydepending upon the host treated and the particular mode ofadministration.

The solid dosage forms for oral administration including capsules,tablets, pills, powders, and granules noted above comprise one or moretypes of particle of the present discovery admixed with at least oneinert diluent such as sucrose, lactose, or starch. Such dosage forms canalso comprise, as in normal practice, additional substances other thaninert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets, and pills, the dosage forms can alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration can include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions can also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavoring, andperfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or setting agents and suspending agents. Thesterile injectable preparation can also be a sterile injectable solutionor suspension in a nontoxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that can be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil can be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Pharmaceutically acceptable carriers encompass all the foregoing and thelike.

As those of ordinary skill in the art will recognize, after reading thisspecification the foregoing discussion is also applicable to the use ofparticles as described herein, wherein such particles comprise apharmaceutically active agent intended to be self delivered to a site inthe body.

Treatment Regimen

The dosage regimen to prevent, give relief from, or ameliorate a diseasecondition, is selected in accordance with a variety of factors. Theseinclude the type, age, weight, sex, diet, and medical condition of thepatient, the severity of the disease, the route of administration,pharmacological considerations such as the activity, efficacy,pharmacokinetics and toxicology profiles of the particular particle orparticle/pharmaceutically active agent combination employed, whether adrug delivery system is utilized, and whether the particles areadministered as part of a drug combination. Thus, the dosage regimenactually employed can vary widely and therefore deviate from thepreferred dosage regimen illustratively set forth above.

Detectable Emitting Fluorescence Labeling

Various useful fluorescent compounds may be successfully employed tolabel the polymer nano-conjugate including using fluorescing moieties,such as green fluorescent proteins, organelle-specific dyes and ionindicators, or a combination of fluorescing markers (for example afluorochrome with green emission light for one moiety and anotherfluorochrome with blue emission for a different moiety).

Fluorescent moieties such as proteins may be employed as a fluorescingmarker if desired and would be visualized through a microscope withappropriate light source or quantified by exposing to a suitable lightsource and determining fluorescence by a spectrofluorometer.

In an aspect, the fluorescence signal from the probe is expresseddirectly as the emission of GFP or any other fluorescent proteinattached to the probe when the fluorescent protein is excited at anappropriate wavelength of bombarded light. In an aspect, a functionalimmunoprobe produces a discernible, detectable and measurablefluorescence signal (or luminescence signal), an image (of capturedfluorescence) which is competently reliably and accurately captured byvisual inspection aided by a microscope or acquired by appropriatecamera and computer software to be displayed visually on a computermonitor for a person for viewing. The intensity and duration of thefluorescence signal is detectable and is reproducible. The images offluorescing cells may be projected on a monitor and compared to anotherimage of a standard. A person can then visually compare such images andmake a determination on whether there is a difference between the imagescompared.

As used herein the term “fluorescent protein” refers to any protein thatis genetically encoded and expressed as a fusion with a wild type ormutant subunit type such that it emits a fluorescent signal that isdetectable using appropriate methods when excited at the necessarywavelength of light.

As used herein, the term “GFP” refers to the Green Fluorescent Proteinfrom Aequorea victoria.

In an aspect, useful nonlimiting illustrative fluorescent proteinsinclude modified green fluorescent proteins including but not limited tothose disclosed in U.S. Pat. No. 6,319,669 which issued to Roger Tsienon Nov. 20, 2001, Wavelength Engineering Fluorescent Proteins, ModifiedGreen Fluorescent Proteins as disclosed in U.S. Pat. No. 5,625,048 whichissued to Roger Tsien on Apr. 29, 1997 and Modified Green FluorescentProteins as disclosed in U.S. Pat. No. 5,777,079 which issued to RogerTsien on Jul. 7, 1998. See http://www.uspto.gov/patft/index.html in thisregard.

Advantageously because of the selective reactivity of the SCK shell orcore and the hydrophobicity of the SCK core, many fluorophores (innumber and type) may be are attached or sequestered if desired into theSCK nanostructure.

Sequence Listings

SEQ ID NO. 1

PTD Amino Acid Sequence—protein transduction domain of HIV-1 TAT (Yoonet al., J. Microbiol., 2004, 42(4), 328-335):Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg^(a)

Note: ^(a)Gly-Arg-Lys-Lys-Arg-Arg is a potential nuclear localizationsequence. Abbreviation: PTD, protein-transduction domain.

SEQ ID NO. 2

Sequence of the unr mRNA (from the GenBank database: Homo SapiensNRAS-related gene (UNR) mRNA) with the PNA50 binding site underlined. 1gcttatggcg gcgctggaga gggggcgctg agctgttggg tatgaagtgt aacagaacag 61actttaccac ctgaaactgc tgcttcaagt tcagatcagg caaggaacaa acctcgtaac 121aactaacaag accaaagaag agtacactta agttgaagac acaacacttg atctgaaaca 181agaagtttgt gcctactcaa cagctttgaa agagcacttc ccaacgctgc tagtagtctt 241tgttttcttc agtgctgtac tgtgagattg cccggtacag cagcagttgt attctttatt 301agcttggtag atcattttct ctcgctcttt tttttaatac tagcaacttt catcctttga 361aacgtgtgct gaaaaagaag aatcagcaaa tactactgaa agtgcaatat ttgagtatca 421ctgcgagatg agctttgatc caaaccttct ccacaacaat ggacataatg ggtaccctaa 481tggtacttca gcagcactgc gtgaaactgg ggttattgaa aaactgttaa cctcttacgg 541atttattcag tgttcagaac gtcaagctag acttttcttc cactgttcac agtataatgg 601caacctgcaa gacttaaaag taggagatga tgttgaattt gaagtatcat cggaccgacg 661gactgggaaa cccattgctg ttaaactggt gaagataaaa caagaaatcc tccctgaaga 721acgaatgaat ggacaagttg tgtgcgctgt tcctcacaac ttagagagta aatctccagc 781tgccccgggt cagagtccaa cagggagtgt atgctacgaa cgtaatgggg aagtgtttta 841tctgacttac acccctgaag atgtcgaagg gaacgttcag ctggaaactg gagataaaat 901aaactttgta attgataaca ataaacatac tggtgctgta agtgctcgca acattatgct 961gttgaaaaag aaacaagccc gctgtcaggg agtagtttgt gccatgaagg aggcatttgg 1021ctttattgaa agaggtgatg ttgtaaaaga gatattcttt cactatagtg aatttaaggg 1081tgacttagaa accttacagc ctggcgatga tgtggaattc acaatcaagg acagaaatgg 1141taaagaagtt gcaacagatg tcagactatt gcctcaagga acagtcattt ttgaagatat 1201cagcattgaa cattttgaag gaactgtaac caaagttatc ccaaaagtac ccagtaaaaa 1261ccagaatgac ccattgccag gacgcatcaa agttgacttt gtgatcccta aagaacttcc 1321ctttggagac aaagatacga aatccaaggt gaccctgctg gaaggtgacc atgttaggtt 1381taatatttca acagaccgac gtgacaaatt agagcgagca accaatatag aagttctgtc 1441aaatacattt cagttcacta atgaagcccg agaaatgggt gtgattgctg ccatgagaga 1501tggttttggt ttcatcaagt gtgtggatcg tgatgttcgt atgttcttcc acttcagtga 1561aattctggat gggaaccagc tccatattgc agatgaagta gagtttactg tggttcctga 1621tatgctctct gctcaaagaa atcatgctat taggattaaa aaacttccca agggcacggt 1681ttcatttcat tcccattcag atcaccgttt tctgggcacg gtagaaaaag aagccacttt 1741ttccaatcct aaaaccacta gcccaaataa aggcaaagag aaggaggctg aggatggcat 1801tattgcttat gatgactgtg gggtgaaact gactattgct tttcaagcca aggatgtgga 1861aggatctact tctcctcaaa taggagataa ggttgaattt agtattagtg acaaacagag 1921gcctggacag caggttgcaa cttgtgtgcg acttttaggt cgtaattcta actccaagag 1981gctcttgggt tatgtggcaa ctctgaagga taattttgga tttattgaaa cagccaatca 2041tgataaggaa atctttttcc attacagtga gttctctggt gatgttgata gcctggaact 2101gggggacatg gtcgagtata gcttgtccaa aggcaaaggc aacaaagtca gtgcagaaaa 2161agtgaacaaa acacactcag tgaatggcat tactgaggaa gctgatccca ccatttactc 2221tggcaaagta attcgccccc tgaggagtgt tgatccaaca cagactgagt accaaggaat 2281gattgagatt gtggaggagg gcgatatgaa aggtgaggtc tatccatttg gcatcgttgg 2341gatggccaac aaaggggatt gcctgcagaa aggggagagc gtcaagttcc aattgtgtgt 2401cctgggccaa aatgcacaaa ctatggctta caacatcaca cccctgcgca gggccacagt 2461ggaatgtgtg aaagatcagt ttggcttcat taactatgaa gtaggagata gcaagaagct 2521ctttttccat gtgaaagaag ttcaggatgg cattgagcta caggcaggag atgaggtgga 2581gttctcagtg attcttaatc agcgcactgg caagtgcagc gcctgtaatg tttggcgagt 2641ctgtgagggc cccaaggctg ttgcagctcc tcgacctgat cggttggtca atcgcttgaa 2701gaatatcact ctggatgatg ccagtgctcc tcgcctaatg gttcttcgtc agccaagggg 2761accagataac tcaatggggt ttggtgcaga aagaaagatc cgtcaagctg gtgtcattga 2821ctaaccacat ccacaaagca caccattaat ccactatgat caagttgggg ggaatctggt 2881gaagggttct gaatatctcc ctcttcatcc ctcccgaaat ctggaatact tattctattg 2941agctattaca ccagttttaa caccttcctc gtgttatgtt taaaaaaata aataaattta 3001agaaaaccat tttaaataat gcacagttgc agcctggaaa aacttaaggt ggcgccttat 3061agtatcaatt ttaggagctt tatttggtgc atttaacgca actggtaatt gcagaatcca 3121ctttgcctgt gtaagtgaaa aatatagact gttatcttgt tggccctatg aaattctgca 3181cttttcatta tatactctac cttcattaat tacttctggc aagatgttct gccttagcac 3241tcagttgcat tcttttcctt tttcttcctg ttcattatgc tttaattctg aggaccatat 3301gagggtagaa tatattatct tttaaaaatt acaaaaattt gtataggcaa accatttctt 3361aaagttgatg gccaaatttt aaaatgttat ttttcatatc atttataatc ttgtcacaat 3421ccacttaaag aagtttggtt atatttcagt gaaaattttc ttccagagta ggtttttttt 3481cgtgggttgg ggggtaactt tactacaatt agtaagtatg gtgcagaatt tcatgcaaat 3541gaggagtgcc agcagtgtga taatttaaac atatttaaac aaaaacaaaa aaaatgaatg 3601cacaaacttg ctgctgctta gatcactgca gcttctagga cccggtttct tttactgatt 3661taaaaacaaa acaaaaaaaa ataaaaaagt tgtgcctgaa atgaatcttg ttttttttta 3721taagtagccg cctggttact gtgtcctgta aaatacagac acttgaccct tggtgtagct 3781tctgttcaac tttatatcac gggaatggat gggtctgatt tcttggccct cttcttgaat 3841tggccatata cagggtccct ggccagtgga ctgaaggctt tgtctaagat gacaagggtc 3901agctcagggg atgtggggga gggcggtttt atcttccccc ttgtcgtttg aggttttgat 3961ctctgggtaa agaggccgtt tatctttgta aacacgaaac atttttgctt tctccagttt 4021tctgttaatg gcgaaagaat ggaagcgaat aaagttttac tgatttttga gacact SEQ ID NO.3 PNA50: TGGTGTGCTTTGTGGATG SEQ ID NO. 4 PNA50S: CATCCACAAAGCACACCA

The following non-limiting examples illustrate various aspects of thepresent discovery.

EXAMPLE Sets A-F hereinafter shows aspects of this discovery.

EXAMPLE Set A shows preparation of SCK nanoparticles

EXAMPLE Set B shows peptide-derivatized SCK-cross-linked nanoparticlesalong with synthesis and characterization illustrating preparing ofpeptide permeation associated with a biologically active particle asdisclosed in Becker et al. (Bioconjug. Chem., 2004, 15, 699-709) whichis incorporated herein in its entirety by reference.

EXAMPLE Set C shows peptide-derivatized shell-cross-linked nanoparticlesand associated biocompatibility evaluation as disclosed in Becker et al.(Bioconjug. Chem., 2004, 15, 710-717) which is incorporated herein inits entirety by reference.

EXAMPLE Set D shows microPET imaging of MCF-7 tumor in mice via unrmRNA-targeted peptide nucleic acids as disclosed in Sun et al. (Bioconj.Chem., 2005, 16, 294-305) which is incorporated herein in its entiretyby reference.

EXAMPLE Set E shows targeting MCF-7 cells with antisense PNAs touniquely overexpressed unr mRNA.

EXAMPLE Set F shows microPET imaging of MCF-7 tumor in mice viashell-cross-linked nanoparticles conjugated to unr mRNA-targeted peptidenucleic acids and a permeation peptide.

Abbreviations used in the following examples: “ζ” means zeta potential,“5(6)-FAM SE” means 5(6)-carboxyfluorescein succinimidyl ester, “7-AAD”means 7-aminoactinomysin, “AFM” means atomic force microscopy, “ATCC”means American Type Culture Collection, “ATRP” means atom transferradical polymerization, “Bio-dUTP” means biotinylated deoxyuridinetriphosphate, “BSS” means Earle's balanced salt solution, “cDNA” meanscomplementary DNA, “Chloramine-T” means N-chloro-p-toluensulfonamidesodium salt, “CHO cells” means chinese hamster ovary cells, “DCM” meansdichloromethane, “DIEA” means diisopropylethylamine, “DLS” means dynamiclight scattering, “DMF” means dimethylformamide, “D_(n)” meansnumber-average hydrodynamic diameter, “dNTP” means deoxynucleotidetriphosphate, “DOTA” means1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid, “DSC” meansdifferential scanning calorimetry, “DTPA” meansdiethylenetriaminepentaacetic acid, “D_(v)” means volume-averagediameter, “D_(z)” means intensity-average diameter, “EDTA” meansethylenediaminetetraacetic acid, “ELISA” means enzyme-linkedimmunosorbent assay, “EOB” means end of bombardment, “equiv.” meansequivalent(s), “FACS” means fluorescence activated cell sorting, “FBS”means fetal bovine serum, “FITC” means fluorescein isothiocyanate,“Fmoc” means 9-fluorenylmethoxycarbonyl, “FPLC” means fast proteinliquid chromatography, “FTSC” means fluorescein-5-thiosemicarbazide,“HATU” means O-(7-Azabenzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate, “HBSS” means Hank's balanced salt solution, “HEPES”means 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, “HPLC” meanshigh performance liquid chromatography, “i.p.” means intraperitoneal,“i.v.” means intravenous, “ID” means injected dose, “IgG” meansimmunoglobulin G, “IL1-β” means interleukin-1 beta, “IR” meansinfra-red, “K_(d)” means dissociation constant, “MALDI-TOF” means matrixassisted laser desorption ionization—time of flight, “MALS” means multiangle light scattering, “MEM” means Eagle's minimum essential medium,“M_(n)” means number-average molecular weight, “M_(p)” meanspeak-average molecular weight, “MS” means mass spectrometry, “MTT” means3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide,“M_(w)” means weigh-average molecular weight, “MWCO” means moleculaweight cut-off, “NLS” means nuclear localization sequence, “NMP” meansN-methylpyrrolidone, “NMR” means nuclear magnetic resonance, “NTP” meansnucleotide triphosphate, “ODN” means oligodeoxynucleotide, “p.i.” meanspost injection, “PAA” means poly(acrylic acid), “PAGE” meanspolyacrylamide gel electrophoresis, “PALS” means phase analysis lightscattering, “PBS” means phosphate buffered saline, “PCR” meanspolymerase chain reaction, “PET” means positron emission tomography,“PMA” means poly(methyl acrylate), “PMDETA” meansN-[2-(Dimethylamino)ethyl]N,N′,N′-trimethyl-1,2-ethanediamine, “PNA”means peptide nucleic acid, “PS” means phosphatidylserine, “PTA”phosphotungstinic acid, “PTD” means protein transduction domain (SeeSeq. 1), “r.t.” means room temperature, “RES” means reticulo-endothelialsystem, “ROI” means region of interest, “RT-PCR” means real-timepolymerase chain reaction, “RT-ROL” means reverse transcriptase randomoligodeoxynucleotide library, “s.c.” means subcutaneous, “SAABS” meansserial analysis of antisense binding sites, “SAGE” means serial analysisof gene expression, “SCID” means severe combined immunodeficiency, “SCK”means shell crosslinked nanoparticles, “SDS” means sodium dodecylsulfate, “SE” means sedimentation equilibrium, “SEC” means sizeexclusion chromatography, “SPECT” means single photon emission computedtomography, “SUV” means standard uptake value, “T/B” means tumor toblood ratio, “T/M” means tumor to muscle ratio, “TCPS” means tissueculture polystyrene, “TEM” means transmission electron microscopy, “TFA”means trifluoroacetic acid, “T_(g)” means glass transition temperature,“THF” means tetrahydrofuran, “TIS” means triisopropyl silane, “TNF-α”means tumor necrosis factor alpha, “unr” means upstream of N-ras orN-ras related gene, “UV” means ultra-violet, “v” means partial specificvolume, “δ” means chemical shift.

EXAMPLE SET A

Preparation of SCK Nanoparticles

Poly(tert-butyl acrylate): A 100 mL Schlenk flask that had been ovendried overnight, flame dried under vacuum, and back filled with argonwas charged with copper(I) bromide (891.6 mg, 6.21 mmol). Tert-butylacrylate (38.00 mL, 259.4 mmol),N-[2-(Dimethylamino)ethyl]N,N′,N′-trimethyl-1,2-ethanediamine) (PMDETA)(1.297 mL, 6.21 mmol), and ethyl-2-bromoproprionate (403 μL, 3.10 mmol),were added via argon washed syringes. The solution was degassed by threecycles of freeze-pump-thaw, and following the final thaw cycle themixture was allowed to stir for 10 min before being immersed in an oilbath at 50° C. After 80 min, the oil bath was removed and the reactionvessel was immersed in liquid nitrogen to quench the polymerizationreaction. The reaction mixture was dissolved in tetrahydrofuran (THF),and passed through an alumina plug to remove the metal/ligand catalystsystem. The polymer solution was concentrated and then precipitated andrecovered into cold methanol. The isolated yield was 28.83 g (85%).

Poly(tert-butyl acrylate-b-styrene-d₈): A 100 mL Schlenk flask that hadbeen oven dried overnight, flame dried under vacuum, and back filledwith argon was charged with copper(I) bromide (45.8 mg, 0.32 mmol) andpoly(tert-butyl acrylate) 2 (0.7234 g, 9.77 e⁻² mmol). PMDETA (66.5 μL,0.32 mmol), and styrene-d₈ (3.000 mL, 2.62 mmol), were added via argonwashed syringes. The solution was degassed by three cycles offreeze-pump-thaw, and following the final thaw cycle the mixture wasallowed to stir for 10 min before being immersed in an oil bath at 75°C. After 150 min, the oil bath was removed and the reaction vessel wasimmersed in liquid nitrogen to quench the polymerization reaction. Thereaction mixture was dissolved in THF, and passed through an aluminaplug to remove the metal/ligand catalyst system. The polymer solutionwas concentrated and then precipitated and recovered into a coldmethanol/water solution. M_(n)=22,500 from SEC, based on MALS.M_(w)/M_(n)=1.06. The isolated yield was 2.0714 g (44%).

Poly(acrylic acid-b-styrene-d₈): The poly(tert-butyl acrylate) block of3 was cleaved selectively by adding 50.00 mL of trifluoroacetic acid(TFA) to 1.0624 g (4.72 e⁻² mmol) of the diblock 2 in 150 mL ofdichloromethane. After 36 hours, the solvent was evaporated in vacuo;the residue was dissolved in THF and purified by dialysis in presoakedcellulose dialysis tubing (12-14 kDa Molecular weight cut-off (MWCO))against nanopure water for 3 days. Lyophilization yielded purepoly(acrylic acid-b-styrene-d₈) recovered as a white powder. Yield:0.8956 g (98%)

Micelle Formation: Spherical micelles of narrow size distribution wereobtained by dissolving the purified block copolymer 4 (0.5186 g, 2.30e⁻⁵ mol) in 250.00 mL of THF (2.07 mg/mL) followed by gradual addition(15.00 mL/h) of an equal volume 5 mM sodium phosphate, 5 mM sodiumchloride, pH 7.4 buffer to induce micellization. The micelles wereallowed to stir for 12 h before being transferred to a dialysis andconcentration cell (10 kDa MWCO) and dialyzed with 3.0 L of buffer. Thefinal volume was 650 mL of buffered micelle solution for a finalconcentration of 0.80 mg/mL.

Shell Crosslinked (SCK) Nanoparticle Formation. Vil21:2,2′-(ethylenedioxy)-bis(ethylamine) (67 μL, 0.46 mmol) was added to0.500 L of micelle solution (0.80 mg/mL) of poly(acrylicacid-b-styrene-d₈). After 30 minutes, an aqueous solution (100 mg/mL) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.040 g,5.46 mmol) was added to the reaction vessel. The reaction mixture wasallowed to stir for 24 h, and the SCK solution was concentrated to 200mL in a concentration cell and washed with 3.0 L of nanopure water toremove the reaction byproducts. A small portion was removed for analysisand the remainder of the SCK solution was lyophilized. (FIG. 2)

Preparation of PNA-Functionalized SCK Nanoparticles

General Procedure for SCK-PNA conjugate formation: To a stirred solutionof SCK (0.53 mg/mL, 2.4 nmoles (particle)) was added Lys-PNA (12 nmoles,5 equiv.) in 0.5 mL water over a 3 minute period. The mixture wasallowed to stir for 30 minutes prior to the addition of 50 equiv. of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (120 nmoles) asa solution in 0.3 mL of water over a 2 minute period. The reaction wasstirred for 16 hours at ambient temperature, and subsequentlytransferred to dialysis tubing (10 kDa MWCO) and allowed to dialyze for4 days against nanopure water. UV-visible spectroscopy demonstrated anaverage of 4 PNAs per particle (80% coupling efficiency).

Characterization of the SCK-PNA Conjugate Prepared Above

Equimolar amounts of SCK and SCK-PNA conjugate were analyzed byUV-visible spectroscopy to determine the concentrations of PNA withinsolution, by subtracting the measured absorbance of the SCK at 260 nmfrom the measured absorbance at 260 nm for the PNA-SCK conjugate, andthe molarity of PNA within solution was determined using a molarextinction coefficient of 150,000 M⁻¹ cm⁻¹ (See FIG. 4). Successfulconjugation of the PNA and loss of non-coupled PNA was determined bysedimentation velocity analysis. The PNA-SCK conjugate was centrifugedat 25,000 rpm and the meniscus demonstrated a loss of absorbance at 260nm indicating a lack of free PNA within the solution (See FIG. 5).

Preparation of SCK-PNA-FTSC: (Fluorescent Labeling)

To a stirred solution of the aforementioned SCK-PNA (1.8 mL, 0.53 mg/mL,1.7 nmoles (particle)) was added Fluorescein-5-thiosemicarbazide (FTSC)(8.5 nmoles, 7.4 μg, 5 equiv. per particle) in 0.2 mL water over a 5minute period. The mixture was allowed to stir for 30 minutes prior tothe addition of 7.5 equiv. of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (13.6 nmoles,3.9 μg) as a solution in 0.1 mL of water over a 2 minute period. Thereaction was allowed to stir for 16 hours at ambient temperature, andsubsequently transferred to dialysis tubing (10 kDa MWCO) and allowed todialyze for 2 days against nanopure water. Final volume was 2.0 mL,which was subsequently concentrated again to its original volume (1.8mL)

Preparation of PNA- and PTD-Functionalized SCK Nanoparticles andFluorescent Labeling

SCK-PNA-FTSC-PTD: To a stirred solution of SCK-PNA-PTD (1.8 mL, 0.53mg/mL, 1.7 nmoles (particle)) was added PTD (17 nmoles, 0.030 mg, 10equiv. per particle) in 0.3 mL water over a 5 minute period. The mixturewas allowed to stir for 30 minutes prior to the addition of 7.5 equiv.of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (26 nmoles,7.7 μg) as a solution in 0.1 mL of water over a 2 minute period. Thereaction was allowed to stir for 16 hours at ambient temperature, andsubsequently transferred to dialysis tubing (10 kDa MWCO) and allowed todialyze for 3 days against nanopure water.

SCK-PNA-PTD: To a stirred solution of SCK-PNA (9.0 mL, 0.53 mg/mL, 8.6nmoles (particle)) was added PTD from an aqueous solution (190 nmoles,0.35 mg, 22 equiv. per particle). The mixture was allowed to stir for 30minutes prior to the addition of 22 equiv. of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide as a solutionin 0.2 mL of water over a 5 minute period. The reaction was allowed tostir for 16 hours at ambient temperature, after which the reaction wastransferred to dialysis bag (10 kDa MWCO) and dialyzed against water for5 days. The final volume was returned to 8.0 mL via stirred cellultrafiltration.

Preparation of PNA- and PTD-Functionalized SCK Nanoparticles andConjugation with Chelator for Radiolabeling.

SCK-PNA-PTD-DOTA: To a stirred solution of SCK-PNA-PTD (8.0 mL, 0.53mg/mL, 7.8 nmoles (particle)) was added DOTA-NH₂-tri-tert-butyl ester(structure shown in FIG. 6) from a THF solution (400 nmoles, 50 equiv.per particle). The mixture was allowed to stir for 30 minutes prior tothe addition of 50 equiv. of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (119 μg) as asolution in 0.1 mL of water over a 2 minute period. The reaction wasstirred for 16 hours at ambient temperature, after which 1 mL ofconcentrated TFA was added to the solution. The deprotection reactionwas monitored in a separate vial against the small molecule component inan aqueous 10% TFA solution, and was continued until no tert-butylresonance was observed in the ¹H NMR spectrum of the small molecule.After 17 hours, the reaction was subsequently transferred to dialysisbag (10 kDa MWCO) and dialyzed for 5 days against deionized water. Thefinal volume was returned to 8.0 mL via stirred cell ultrafiltration.

FIG. 4 shows UV-visible spectra of SCK and SCK-PNA conjugates. Increasein absorbance at 260 nm demonstrated the concentration of PNAs withinsolution.

FIG. 5 shows a structure for sedimentation velocity analysis ofSCK-PNA50 conjugate. The loss of absorbance at 260 nm at the top of thecell, demonstrated the desired lack of free PNA within the solution.

FIG. 6 shows the DOTA-NH₂-tri-tert-butyl ester chelator used forconjugation to the SCK-PNA-PTD construct.

EXAMPLE SET B

Preparation and Recovery of Peptide-Derivatized Shell-Cross-LinkedNanoparticles

We have demonstrated that our conjugation of the protein transductiondomain (PTD) from the HIV-1 Tat protein to shell-cross-linked (SCK)nanoparticles is an efficacious method to facilitate cell surfacebinding and transduction of SCK nanoparticles (Liu et al., Biomacromol.,2001, 2, 362-368). Attaching increasing numbers of peptide sequences toSCK nanoparticles in a global solution-state functionalization strategyhas been devised as a method for increasing the efficiency of thecell-penetrating process.

In this example, the numbers of peptides per SCK were controlled throughstoichiometric balance and measured by using two independent methods,UV-visible spectroscopy and phenylglyoxal analysis. PTD was conjugatedin 0.005, 0.01, and 0.02 molar ratios, relative to the acrylic acidresidues in the shell, to the SCK nanoparticles resulting in SCKpopulations possessing nominally 52, 104, and 210 (41, 83, and 202 asmeasured by phenylglyoxal analysis) PTD peptides per particle,respectively. The methodologies for the block copolymer and nanoparticlesyntheses, peptide derivatization, and characterization ofpeptide-functionalized SCK nanoparticles are reported and thefeasibility and efficiency of intracellular internalization of therespective SCKs were quantified.

Test Procedures

Materials. Unless otherwise listed, all solvents and reagents werepurchased from Sigma Aldrich Chemical Co. (St. Louis, Mo.) and used asreceived. Monomers were purchased from Sigma Aldrich and distilled overcalcium hydride. Fmoc-protected amino acids and preloaded solid-phaseWang resins were purchased from NovaBiochem-CalBiochem Corp (San Diego,Calif.). Spectra/Por dialysis membranes (Spectrum Laboratories, Inc.,Rancho Dominguez, Calif.) were purchased from Fisher Scientific Company(Pittsburgh, Pa.). Prolong antifade mounting medium was purchased fromMolecular Probes Inc. (Eugene, Oreg.).

Measurements. ¹H NMR (300 MHz) and ¹³C NMR (75 MHz) spectra wererecorded as solutions on a Varian Mercury 300 MHz spectrometer with thesolvent signal as standard. IR spectra were obtained on a Perkin-ElmerSpectrum BX FT-IR system using diffuse reflectance sampling accessories.Mass spectra were obtained from a Voyager DE-RP MALDI-TOF massspectrometer (PE Biosystem). HPLC was performed using a Hewlett-PackardSeries 1100 with reversed phase C18 column (Dynamax, 300 Å, Rainin)equipped with quaternary pump, solvent degasser, and diode arraydetector.

Size Exclusion Chromatography (SEC). Size exclusion chromatography wasconducted on a Model 150-CV (Waters Chromatography Inc., Medford,Mass.). The instrument was equipped with a Model 410 differentialrefractometer, a Model PD2040 dual-angle (15° and 90°) light scatteringdetector (Precision Detectors Inc., Bellingham, Mass.), and athree-column set of gel-mixed-bed styrene-divinylbenzene columns(Polymer Laboratories Inc., Amherst, Mass.). The SEC system wasequilibrated at 35° C. in anhydrous THF, which served as the polymersolvent and eluent (flow rate set to 1.00 mL/min then determinedgravimetrically). An injection volume of 400 μL was used, and thepolymer concentrations ranged from 5 to 10 mg/mL. Data collection wasperformed with program Precision Acquire (Precision Detectors Inc.,Bellingham, Mass.). Data analysis was performed with program Discovery32(Precision Detectors Inc., Bellingham, Mass.). Interdetector delayvolume and the light scattering detector calibration constant weredetermined from a nearly monodisperse polystyrene calibrant (M_(p)=90000g/mol, M_(w)/M_(n)<1.04: Pressure Chemical Co., Pittsburgh, Pa.). Thedifferential refractometer was calibrated with standard polystyrenereference material (SRM 706; NIST, Gaithersburg, Md.), of known specificrefractive index increment, dn/dc (0.184 mL/g). The dn/dc of theanalyzed polymers was then determined from the differentialrefractometer response.

Differential Scanning Calorimetry (DSC). The glass transitiontemperature (T_(g)) was measured by differential scanning calorimetry ona Perkin-Elmer DSC-4 differential scanning calorimeter. Heating rateswere 10° C./min and reported values of T_(g) were measured at themidpoint of the inflection tangent, upon the third heating scan.

Sedimentation Equilibrium (SE). A Beckman Instruments Co. Model OptimaXL-I analytical ultracentrifuge operated with a Model An60-Ti, four-holerotor was used to centrifuge 50 mM NaH₂PO₄, 50 mM NaCl, pH 7.1 solutionsof the SCK to sedimentation equilibrium. All measurements were made at20±0.1° C. Rotor speeds of 2000, 3000, 5000 and 8000 rpm were used.Sedimentation equilibrium data were obtained for three SCK solutionconcentrations. Resulting sedimentation equilibrium profiles wererecorded with the instrument's Rayleigh interferometric (refractiveindex) detection optics. The ultracentrifuge sample cell was assembledfrom an Epon charcoal-filled, six-channel centerpiece and matchedsapphire windows. The solution volume and the cell's optical path lengthwere 110 μL and 12 mm, respectively. The solution volume and opticalpath length employed corresponded to a “short” column sedimenta-tionequilibrium test with a column height of approximately 2.5 mm. Acentrifugation time of 3 to 5 days was used to reach sedimentationequilibrium. Partial specific volume (v) values for the SCKs weredetermined via sedimentation equilibrium analysis for protonated anddeuterated buffer solutions of SCKs as described in Remsen et al.(Macromol., 1999, 32, 3685-3689). Solutions of the SCK in deuteratedbuffer were prepared by exhaustive dialysis of protonated buffer stocksolutions of SCKs against 50 mM phosphate buffered saline (PBS), pD 7.1.In 99.9 atom % D₂O, Calculation of molecular weight employed a built-indata analysis program that employed standard multicomponentleast-squares fitting routines. Density at 20.0±0.1° C. for protonatedand deuterated buffers was determined with a Mettler-Parr digitaldensity meter. Measurements of v were reproducible to within ±1% of themean value given by three determinations. The values of v obtained by SEwere also used for the determination of the weight concentrations of SCKnanoparticle solutions. The digital density meter was employed tomeasure the solution density at 20.0±0.1° C. for the SCK solutions andtheir corresponding PBS to an accuracy of ±0.0001 g/mL.

These values were used in conjunction with the SE-measured value of v todetermine the weight concentration (C_(SCK)) of the SCK solution in PBS:

C_(SCK)=(p_(SCK)−p_(b))/(1−vp_(b)) where p_(b) is the density of thebuffer and p_(SCK) is the density of the SCK solution.

UV-Visible Spectroscopy. Absorption measurements were made using aMolecular Devices Corp (Sunnyvale, Calif.) UV-visible spectrophotometer.A Thermomax multiplate reader using SOFTmax PRO software was employed.The concentrations of PTD and SCK in aqueous solutions of functionalizedSCK nanoparticles were simultaneously determined using the UV absorbancemeasured at 230 and 276 nm. Measurements obtained at 230 and 276 nmcorresponded to the turbidity of the SCK nanoparticle and the tyrosine(TYR) absorbance of the PTD at these wavelengths. The total absorbancefor a functionalized SCK at a given wavelength, A^(λ), was given by thesum of the absorbances of the nanoparticle's SCK component, A_(SCK)^(λ), and its PTD component, A_(TYR) ^(λ):A ²³⁰ =A _(SCK) ²³⁰ +A _(TYR) ²³⁰  Equation (1)A ²⁷⁶ =A _(SCK) ²⁷⁶ +A _(TYR) ²⁷⁶  Equation (2)

The individual concentrations of the SCK and PTD (C_(SCK) and C_(TYR),respectively) components constituting a functionalized nanoparticle wereobtained by simultaneously solving equations 1 and 2 after calibrationof the UV spectrum at 230 and 276 nm with PTD and SCK standards of knownconcentration. The calibration yielded a set of calibrationcoefficients, K, for the components at 230 and 276 nm:A ²³⁰ =K _(SCK) ²³⁰ +C _(SCK) +K _(TYR) ²³⁰ C _(TYR)  Equation (3)A ²⁷⁶ =K _(SCK) ²⁷⁶ +C _(SCK) +K _(TYR) ²⁷⁶ C _(TYR)  Equation (4)

Measured values A²³⁰ and A²⁷⁶ were substituted into equations 3 and 4which were expressed in matrix form (equation 5) and then solved byinversion of the matrix of calibration coefficients, K:A=KC  Equation (5)K ⁻¹ A=K ⁻¹ KC  Equation (6)C=K ⁻¹ A  Equation (7)

Substitution of computed values of K⁻¹ in equation 7 above provided theexpressions used to calculate the weight concentrations of SCK and PTDin solution:C _(SCK)=8.6405A ²³⁰−41.5418A ²⁷⁶  Equation (8)C _(TYR)=9.5652A ²³⁰−1.5869A ²⁷⁶  Equation (9)

Testally determined weight concentrations were converted to molarconcentrations using the known molecular weight of the PTD and themeasured molecular weight of the SCK. Molar concentrations of eachcomponent provided the requisite information to evaluate the number ofpeptides (PTD) per SCK nanoparticle (no. peptides per SCK=C_(PTD)(M)/C_(SCK) (M)).

Phenylglyoxal Analysis Measurements. The number of arginine residues(and PTD) was determined using phenylglyoxal analysis. The assay wascalibrated by adding aliquots (100 μL) of a PTD solution (100 μL in PBSwith 10% methanol, volume fraction) in row 1 of a quartz 96-well plateand diluting serially through column 11. SCK solutions (100 μL) (0.0,0.5, 1.0, and 2.0%) of known concentrations (1.20, 2.00, 1.20, and 0.60mg/mL. respectively) were added to each well in the first column anddiluted serially as described above. An aliquot (100 μL) of aphenylglyoxal solution (600 μM), in identical buffer, was then added toeach well of the plate, and the reaction was allowed to incubate at 4°C. overnight. The 96-well plate was read for absorbance at λ=310 nm ineach well. The control phenylglyoxal absorbance spectrum was subtractedfrom the PTD absorbance spectra, and by plotting the residual absorbancenumbers vs the known concentration of PTD in each well, a calibrationcurve was generated from which the amount of PTD in unknown solutionscould be determined. Measurement of the SCK concentrations by othermethods and converting molar ratios provided quantitative information onthe number of peptides per particle in each of the four samples.

Dynamic Light Scattering (DLS). Hydrodynamic diameter distribution anddistribution averages for the SCKs in PBS solution were determined bydynamic light scattering. A Brookhaven Instruments Co. (Holtsville,N.Y.) DLS system equipped with a Model BI-9000AT digital correlator, aModel EMI-9865 photomultiplier, and a Model 95-2 Ar ion laser (LexelCorporation, Fremont, Calif.), operated at 514.5 nm, was used.Measurements were made at 20±0.1° C. Nanoparticles were dialyzed into 50mM PBS, pH 7.1, prior to analysis. Buffered nanoparticle solutions wereeither centrifuged in a model 5414 microfuge (Brinkman InstrumentCompany, Westbury, N.Y.) for 4 min or filtered through 0.22 μmpoly(vinylidene fluoride) and 0.1 μm ceramic filters (Whatman,Maidstone, UK) to remove dust particles. Scattered light was collectedat a fixed angle of 90°. The digital correlator was operated with 522ratio spaced channels, an initial delay of 1.6 μs, a final delay of 10ms, and a duration time of 15 min. A photomultiplier aperture of 400 μmwas used, and the incident laser power was adjusted to obtain a photoncounting rate between 200 and 300 Kcps. Only measurements for which themeasured and calculated baselines of the intensity autocorrelationfunction agreed to within ±0.1% were used to calculate nanoparticlehydrodynamic diameter values. All determinations were made intriplicate. The calculations of the nanoparticle diameter distributionsand distribution averages were performed with the ISDA software package(Brookhaven Instruments Co, Brookhaven Instruments Limited, ChapelHouse, Stock Wood, Redditch, Worcestershire, B96 6ST, UK), whichemployed single-exponential fitting, cumulants analysis, andnon-negatively constrained least-squares particle size distributionanalysis routines.

Zeta Potential. Zeta potential (ζ) values for the SCKs were determinedwith a Brookhaven Instrument Co. (Holtsville, N.Y., USA) Model ZetaPluszeta potential analyzer. Measurements were made following dialysis (MWCO12-14 kDa dialysis tubing, Spectrum Laboratories, Rancho Dominguez,Calif., USA) of SCK solutions into 1 mM KH₂PO₄, 1 mM KCl, pH 7.1 buffer.Data were acquired in the phase analysis light scattering (PALS) mode,following solution equilibration at 25° C. Calculation of ζ from themeasured nanoparticle electrophoretic mobility (μ) employed theSmoluchowski equation: μ=∈ ζ/η where ∈ and η are the dielectric constantand the absolute viscosity of the medium, respectively. Measurements ofζ were reproducible to within ±2 mV of the mean value given by 16determinations of 10 data accumulations.

Transmission Electron Microscopy (TEM). Transmission electron microscopycarbon grids were prepared by oxygen plasma treatment to make thesurface hydrophilic. Particle samples were diluted 9:1 in water andfurther diluted 1:1 with a 1% (mass fraction) phospho-tungstinic acid(PTA) stain. Micrographs were collected at 100000 magnifications andcalibrated using a 41 nm polyacrylamide bead from NIST. Histograms ofparticle diameters were generated from the analyses of a minimum of 150particles from at least three different micrographs.

Atomic Force Microscopy (AFM). Tapping-mode atomic force microscopymeasurements were conducted in air with a Nanoscope III Bioscope system(Digital Instruments, Santa Barbara, Calif.) operated under ambientconditions with standard silicon tips (OTESPA-70; L, 160 μm; normalspring constant, 50 N/m; resonance frequency, 246-282 kHz). The sampleswere prepared for AFM analysis by depositing a 2-μL drop of the solutiononto freshly cleaved mica and allowing it to dry freely in air.Histograms of particle heights were generated from the section analysisof a minimum of 150 particles from at least five different analysesregions. Cell Lines. Chinese hamster ovary (CHO) cells (ATCC, AmericanType Culture Collection, Manassas, Va.) were obtained from WashingtonUniversity in St. Louis. CHO cells were maintained in RPMI 1640 (LifeTechnologies, Rockville, Md.) culture medium supplemented with 10%(volume fraction) heat-inactivated fetal bovine serum (FBS, LifeTechnologies, Rockville, Md.), in 5% CO₂:95% air (volume fractions) at37° C.

Transduction Tests. CHO cells were cultured, counted, and resuspended toa final concentration of 100,000 cells/mL. An aliquot of the cellsuspension (3.00 mL) was deposited into each well of a tissue culturetreated six-well plate (Falcon, 3043), which contained a No. 1.5 glasscover slip (Corning). After 48 h, the cells (50-60% confluence) in eachsix-well plate were washed with PBS (2×5.00 mL). An aliquot of serumfree RPMI 1640 medium (3.00 mL) was added to each well followed by therespective SCK solutions (1.00 mL each). The plates were then returnedto the incubator to incubate at 37° C. After 1 h, the nanoparticles wereremoved, and each well was washed with PBS (3×5.00 mL). The cells werethen fixed using a 4% (mass fraction) paraformaldehyde solution (2.00mL) in each well and allowing ambient temperature incubation for 1 h.After fixation, the cells were mounted in Prolong antifade mountingmedium (Molecular Probes, Eugene, Oreg.) and viewed under bright fieldand fluorescent conditions using an Olympus IX-70 inverted microscope.The transduction tests were repeated using RPMI 1640 medium supplementedwith 0.1% (mass fraction) sodium azide, and at 4 and 37° C. Forquantification by flow cytometry, the transduction tests were repeatedexcept that the cells were grown directly on the tissue cultured platesinstead of glass cover slips. The cells were trypsinized to remove mostof the surface bound particles and release the cells from the plate,centrifuged at 4° C., and resuspended in PBS and stored on ice briefly.Less than 5 min prior to flow cytometry analysis, propidium iodide (5.0μL) was added to each centrifuge tube. Analysis was done on a FACSCalibur (BD Biosciences, Franklin Lakes, N.J.) instrument usingCellQuest software.

Fluorescence and Confocal Microscopy. Confocal microscopy wasaccomplished using a Leica TCS SP2 scanning confocal microscopy systemequipped with Ar⁺, Kr⁺, and HeNe laser systems. The excitationwavelength was 488 nm, and the emission was collected over the range of510-530 nm. Optical slices were taken on 0.5 μm centers with the Leicasoftware, compiling the collections of images into the 3-Dreconstructions. The incubation and fixation testal parameters outlinedpreviously were strictly adhered to in the confocal microscopy testswith the following exceptions. The cover slips from the 37° C. testswere mounted on slides fitted with a small spacer and PBS was added asthe medium. These tests were repeated with cells being incubated at 4°C. for 1 h and washed and mounted with cold buffer. The cells wereviewed for a maximum of 20 min as live whole, unfixed specimens.

Fmoc-Solid-Phase Synthesis of PTD on Resin. The protein transductiondomain sequence (GGGGYGRKKRRQRRR) was synthesized by standardsolid-phase synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry.A small amount of beads were removed and washed with N-methylpyrrolidone(NMP), dimethylformamide (DMF), CH₂Cl₂, and methanol three times each.Peptide cleavage was achieved through treatment of the resin with a 10mL 95% TFA: 2.5% triisopropyl silane (TIS): 2.5% water solution (volumefractions) for a minimum of 4 h. The solution was filtered, and thebeads were rinsed with TFA. The solution was concentrated in vacuo, andthe concentrate was precipitated into cold ether. The precipitates wereeffectively centrifuged at 3500 rpm for 10 min. The supernatant wasdecanted, the pellet was resuspended in cold ether, and thecentrifugation process was repeated. The pellet was purified andrecovered by reversed phase HPLC. MS (MALDI): 1789.5022 [M+H]⁺ (calcd:1788.03 MW). TFA is C₂HF₃O₂, available from AppliChem GmbH, Ottoweg 4,D-64291 Darmstadt, Germany

Poly(tert-butyl acrylate) (2). A 100 mL Schlenk flask that had beenoven-dried overnight, flame-dried under vacuum, and back-filled withargon was charged with copper(I) bromide (891.6 mg, 6.2×10⁻³ mol).tert-Butyl acrylate (38.00 mL, 2.6×10⁻¹ mol), PMDETA (1.30 mL, 6.2×10⁻³mol), and ethyl-2-bromopropionate (403 μL, 3.1×10⁻³ mol) were added viaargon-washed syringes. The solution was degassed by three cycles offreeze-pump-thaw, and following the final thaw cycle, the mixture wasstirred for 10 min before being immersed in an oil bath at 50° C. After80 min, the polymerization was quenched by immersion in liquid nitrogen.The reaction mixture was dissolved in THF and passed through an aluminaplug to remove the metal/ligand catalyst system. The polymer solutionwas concentrated and the product recoved by being precipitated into coldmethanol. M_(n) 7400 from SEC. based on MALS. M_(w)/M_(n)=1.12. Therecovery yield of the isolated product was 28.83 g (85%).(T_(g))_(tBA)=33° C. IR: 3440, 2980. 1750, 1450. 1380, 1265, 1150, 850,760, 625 cm⁻¹. ¹H NMR (CDCl₃) δ 1.05 (d, CH₃CH end group), 1.22 (t,CH₃CH₂O end group), 1.20-1.50 (broad, (CH₃)₃C), 1.24-1.70 (broad, mesoand racemo CH₂ of the polymer backbone), 1.74-1.94 (broad, meso CH₂ ofthe polymer backbone), 2.15-2.35 (broad, CH of the polymer backbone),4.05 (br overlapping m, CH₃CH₂O and CHBr end groups) ppm. ¹³C NMR(CDCl₃,) δ 27.9-28, 35.5-37.4, 41.5-41.4, 80.3, 173.6, 173.9 ppm.

Poly(tert-butyl acrylate-b-methyl acrylate) (3). A Schlenk flask (100mL) was oven-dried overnight, flame-dried under vacuum, back-filled withargon, and charged with copper I bromide (184.1 mg, 1.3×10⁻³ mol) andpoly(tert-butyl acrylate) 2 (4.7381 g, 6.4×10⁻⁴ mol). PMDETA (268 μL,1.3×10⁻³ mol) and methyl acrylate (30.00 mL, 3.3×10⁻¹ mol) were addedvia argon-washed syringes. The solution was degassed by three cycles offreeze-pump-thaw, and following the final thaw cycle, the mixture wasstirred for 10 min before being immersed in an oil bath at 68° C. After70 min, the polymerization was quenched by immersion in liquid nitrogen.The reaction mixture was dissolved in THF and passed through an aluminaplug to remove the metal/ligand catalyst system. The polymer solutionwas concentrated and then precipitated into a cold methanol/watersolution. M_(n)=23400 from SEC, based on MALS. M_(w)/M_(n)=1.13. Theyield was 9.18 g (16%). (T_(g))_(PtBA)=32° C. (T_(g))_(PMA)=14° C. IR:3650. 3450, 2970, 1990, 1550, 1450, 1180, 1080, 850 750. 625 cm⁻¹. ¹HNMR (CDCl₃) δ 1.20-1.50 (broad, (CH₃)₃C), 1.25-1.95 (broad, CH₂ of thepolymer backbone), 1.74-1.94 (broad, CH₂ of the polymer backbone),2.15-2.40 (broad, CH of the polymer back-bone), 3.55-3.65 (broad. OCH₃,)ppm. ¹³C NMR (CDCl₃) δ 27.9-28, 35.5-37.4, 41.5-41.4, 51.5, 80.3, 173.4,173.6 ppm.

Poly(acrylic acid-b-methyl acrylate) (4). The tert-butyl esters alongthe poly(tert-butyl acrylate) block of 3 were cleaved selectively byadding TFA (30.00 mL) to the diblock, 3 (8.5600 g. 3.66×10⁻⁴ mol), indichloromethane (100 mL). After 36 h, the solvent was evaporated invacuo, and the residue was dissolved in THF and purified by dialysis inpresoaked dialysis tubing (MWCO 12-14 kDa) against Nanopure (18.0 mΩcm⁻¹) water for 3 days. Lyophilization yielded poly(acrylicacid-b-methyl acrylate) as a white powder. Yield: 7.25 g (98%);(T_(g))_(PAA)=145° C. (T_(g))_(PMA)=14° C. IR: 3500-2500, 1760, 1660,1445. 1280, 1180, 1050, 850, 725, 610 cm⁻¹. ¹H NMR (CDCl₃) δ 1.25-2.0(broad, CH₂ of the polymer backbone), 1.74-1.94 (broad, CH₂ of thepolymer backbone), 2.20-2.45 (broad, CH of the polymer backbone),3.55-3.65 (broad. OCH₃,) ppm. ¹³C NMR (CDCl₃,) δ 34-36.4, 40.5-42.4,50.5, 173, 178 ppm.

Micelle Formation. Polymer micelles of narrow size distribution wereobtained by dissolving the block copolymer 4 (1.9968 g, 8.53×10⁻⁵ mol)in THF (1.000 L, 1.997 mg/mL) followed by gradual addition (20.00 mL/h)of an equal volume of nonsolvent (H₂O) for the hydrophobic poly(methylacrylate) to induce micellization. The micelles were stirred for 12 hbefore being transferred to presoaked and rinsed dialysis bags (MWCO12-14 kDa) and dialyzed against Nanopure (18.0 MΩ cm⁻¹) water for 3 daysto remove the organic solvent. The final volume was 2.250 L of aqueousmicelle solution for a final concentration of 0.89 mg/mL. DLS: D_(h)=38nm, ZETA: ζ=−27±0.7 mV, TEM: 21.3±3.8 nm.

Shell-Crosslinked (SCK) Nanoparticle Formation.2,2′-(Ethylenedioxy)-bis(ethylamine) (192 μL, 1.3×10⁻³ mol) was added to2.125 L of micelle solution (0.90 mg/mL) of poly(acrylic acid-b-methylacrylate). After 30 mm, an aqueous solution of1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.0201 g,1.05×10⁻² mol) was added to the reaction vessel. The reaction mixturewas stirred for 24 h, and it was then dialyzed.

FIG. 11 shows sequential atom transfer radical polimerization (ATRP) oftert-butyl acrylate and methyl acrylate afforded well-definedpoly(tert-butyl acrylate-b-methyl acrylate)

The tert-butyl esters were cleaved selectively through treatment of thediblock copolymer with TFA in CH₂Cl₂ for 36 h, against Nanopure waterfor 3 days to remove residuals. The SCKs were dialyzed into 50 mM sodiumphosphate. 50 mM sodium chloride, pH 7.4 buffer, and the number-averagehydrodynamic diameter (D_(n)) of were determined by dynamic lightscattering. DLS: D_(n)=37 nm. ZETA: ζ=−20±0.5 mV. TEM: 18.3±3.6 nm. AFM:3.8±1.8 nm. IR: 3385, 2949, 1738, 1568, 1442, 1163, 829 cm⁻¹. DSC:T_(g)=15° C. ¹H NMR (D₂O:THF-d₈, 1:2 (volume fraction)) δ 1-3.0(aliphatic protons of polymer backbone), 3.50-3.70 (OCH₃) ppm.

General procedure for global solution state functionalization of (SCK)nanoparticles with various molar ratios of PTD. An SCK solution (50.00mL, 0.895 mg/mL) was placed into a 100 mL round-bottom flask equippedwith a stir bar. Sodium chloride (1.00 g) was placed into each of theflasks to minimize aggregation. A stock solution (50.00 mg/mL) of1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in sodiumphosphate buffer, pH 7.4 was prepared, and 1.00 mL aliquots (2.5×10⁻⁴mol) were added to each flask to activate the acrylic acid residues. Astock solution (18.21 mg/mL) of the PTD peptide was made in sodiumphosphate buffer, pH 7.4, and aliquots of the solution were added to therespective flasks 20 min after the addition of the carbodiimide.

0.5% PTD Functionalized. A PTD stock solution (61 μL, 18.21 mg/mL,6.25×10⁻⁷ mol) was added to the activated SCK solution, and the mixturewas allowed to react overnight. Following the allocated reaction time,the solution was transferred to presoaked dialysis tubing (MWCO 12-14kDa) and allowed to dialyze for 3 days against 50 mM sodium phosphate,50 mM sodium chloride, pH 7.4 buffer, prior to analysis. DLS: D_(n)=35±3nm. ZETA: ζ=−28±0.9 mV. TEM: 20.2±3.7 nm.

1.0% PTD Functionalized. A PTD stock solution (123 μL, 18.21 mg/mL,1.25×10⁻⁶ mol) was added to the activated SCK solution, and the mixturewas allowed to react overnight. Following the allocated reaction time,the solution was transferred to presoaked dialysis tubing (MWCO 12-14kDa) and allowed to dialyze for 3 days against 50 mM sodium phosphate,50 mM sodium chloride, pH 7.4 buffer, prior to analysis. DLS: D_(n)=36±3nm. ZETA: ζ=−23±0.7 mV. TEM: 21.4±3.6 nm.

2.0% PTD Functionalized. A PTD stock solution (245 μL, 18.21 mg/mL,2.50×10⁻⁶ mol) was added to the activated SCK solution, and the mixturewas allowed to react overnight. Following the allocated reaction time,the solution was transferred to presoaked dialysis tubing (MWCO 12-14kDa) and allowed to dialyze for 3 days against 50 mM sodium phosphate,50 mM sodium chloride, pH 7.4 buffer, prior to analysis. DLS: D_(n)=32±3nm. ZETA: ζ=−22±0.5 mV. TEM: 21.6±3.3 nm.

General procedure for the conjugation of a fluorescent tag to thePTD-functionalized nanoparticles. The respective PTD-functionalized SCKsolutions (10.00 mL. 0.895 mg/mL) (2.24×10⁻⁹ mol) were placed into 50 mLround-bottom flasks. A 4.0 M KCl solution (1.00 mL) was added to eachflask. The vessels were allowed to equilibrate for 30 min before 0.500mL of a 50 mg/mL carbodiimide stock solution (1.25×10⁻⁴ mol) was addedto each vessel. After 15 min, 200 μL of a 0.400 mg/mL stock solution(1.58×10⁻⁷ mol) of the fluorescein derivative was added to eachround-bottom flask, and the reactions were stirred overnight. Thesolutions were then transferred to successfully presoaked dialysis bags(MWCO 12-14 kDa) and allowed to dialyze for 3 days against 50 mMphosphate, 50 mM NaCl buffer at pH 7.4. The reaction resulted inapproximately 70 fluorophores being coupled to each nanoparticle basedon calculations assuming ideal reaction efficiency.

Results and Discussion

SCK Synthesis and Peptide Derivitization. The amphiphilic blockcopolymer precursor to the SCKs was prepared by sequential atom transferradical polymerizations of tert-butyl acrylate and methyl acrylatefollowed by the selective acidolysis of the tert-butyl esters. Thepoly(tert-butyl acrylate) macroinitiator was afforded through thepolymerization of tert-butyl acrylate at 50° C. for 80 min using ethyl2-bromopropionate as the initiator and the Cu^(I)Br/PMDETA catalyticsystem. The chain was further extended through the additionpolymerization of methyl acrylate in bulk in the presence ofCu^(I)Br/PMDETA at 68° C. for 70 min as shown in FIG. 11. By allowingthe reaction to proceed under conditions of high dilution (in monomer),low conversion, and by using a liquid nitrogen quench, a narrowmolecular weight distribution for the block copolymer was maintained.The tert-butyl esters were cleaved selectively upon reaction with TFA inCH₂Cl₂. The solvation of the purified poly(acrylic acid-b-methylacrylate) block copolymer in tetrahydrofuran (1.99 mg/mL) followed bythe controlled addition of Nanopure (18 MΩ cm) water (20.00 mL/h), andextensive dialysis against deionized water afforded micelles.

SCKs were formed by intramicellar conversion of approximately 50% of the1-[3′-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride activatedacrylic acid residues in the shell layer to amides using2,2′-(ethyl-enedioxy)bis(ethylamine), as shown in process FIG. 12. Theprotein transduction domain sequence of HIV-1 Tat protein extended byfour glycine residues (GGGGYGRKKRRQRRR) used in this study waschemically synthesized by Fmoc solid-phase peptide methodologies. Thefour additional glycine residues were placed at the N-terminus tofunction as a spacer between the active peptide sequence and themacromolecular assembly. The peptide was cleaved from the support,purified by reverse phase HPLC, and then coupled in 0.005, 0.01 and 0.02molar ratios, relative to the acrylic acid residues within the SCKnanoparticles. The peptide coupling to the SCKs involved amidation ofthe N-terminus to the carboxylic acid residues within the SCK shell. Themolar amounts correspond to approximately 52, 104 and 210 PTD sequencesper particle, respectively, assuming complete conversion and using theaggregation number (185±7) of block copolymers within each particle.Following the conjugation, the respective solutions were then purifiedby dialysis against buffer for 5 days to remove the noncoupled peptidesequences. Non-coupled PTD was identified by UV measurements made atsedimentation equilibrium (SE), as described below.

The SCKs were effectively and successfully functionalized with variousmolar quantities of PTD under high ionic strength conditions to minimizeaggregation and interparticle cross-linking in a global solution statefunctionalization process. A linker-derivatized 5(6)-carboxylfluoresceindye (approximately 70 fluorophores per particle) was coupled to each ofthe SCK samples 6-9 to facilitate observation under fluorescenceconditions.

FIG. 12 shows the SCK (labelled “6” herein) was prepared through themicellization of the block copolymer labelled “4” therein, followed bythe crosslinking of approximately 50% of the acrylic acid residues inthe shell layer with 2,2′-(ethylenedioxy)-bis(ethylamine)^(a)

Molecular Weight and Aggregation Number. The absolute weight-averagemolecular weight, M_(w), of PTD-functionalized SCK nanoparticles wasevaluated by sedimentation equilibrium (SE) analysis. Representative SEdata (fringe displacement versus radial position, r) are presented inFIG. 13(A). The partial specific volume, v, for the SCKs was alsodetermined by SE using a methodology that employed sedimentationequilibrium profiles collected for the SCK dispersed in both protonatedand deuterated buffer. Linear SE plots were obtained by plotting thelog(fringe displacement) versus r²/2 from which the slopes of the plotswere determined and v was computed by use ofv=[k−(σ_(D)/σ_(H))]/[(ρ_(D)−ρ_(H)(σ_(D)/σ_(H))] where ρ_(D) is thedensity of the deuterated buffer, ρ_(H) is the density of the protonatedbuffer, and k is the ratio of the molar masses for the deuterated SCK tothe protonated SCK.

SE analysis of M_(w) employed the nonlinear, least-squares fitting ofsedimentation equilibrium profiles represented by the data in FIG. 13(A)to a single-component model with ρ_(H) and v held constant as measuredparameters. The initial concentration of the nanoparticle at themensicus, c₀, of the sedimentation column, the baseline offset, N₀, andan apparent weight-average molecular weight, M_(w,app), were calculatedas adjustable parameters in the fitting procedure. The computedM_(w,app) when expressed as 1/M_(w,app) and plotted against the initialloading concentration of SCK nanoparticle (FIG. 13(C)) showed adependence on both c and the rotor speed. The observed trends areindicative of molecular weight heterogeneity and nonideal sedimentationdue to interparticle interactions or cross-linking. Extrapolation of1/M_(w,app) to zero concentration for the lowest rotor speed employedprovided the evaluation of M_(w). As summarized in Table 1, the 0%PTD-functionalized SCK nanoparticle produced a v of 0.556±0.007 mL/g,resulting in a M_(w) of 4,000,000±151,000 g/mol. Dividing M_(w) by thecopolymer weight-average molecular weight, corrected for the molar massincrease due to 50% cross-linking of each copolymer chain (664 g/mol)resulted in a weight-average degree of aggregation, N_(w,agg), of 185±7.The absolute weight-average molecular weights, M_(w), of thePTD-functionalized SCK nanoparticles were also evaluated by SE analysis.Interestingly, M_(w) for the 0.5%, 1%, and 2% PTD-functionalized SCKswere 31%, 37%, and 41% higher, respectively, than M_(w) for 0%functionalized SCK. Functional-ization of the preformed SCK (0% PTD)with the peptide was expected to produce small M_(w) increases throughthe incremental addition of molar mass from the PTD. The observedincreases in M_(w) exceed the expected increase by at least 10-fold,suggesting that functionalization with PTD was accompanied by a smallpercentage of internanoparticle cross-linking reactions or electrostaticaggregation events due to the zwitterionic character of thePTD-functionalized nanoparticles.

Collection of SE profiles using UV-visible detection optics allowed theanalysis of the peptide content of the nanoparticles at centrifugationequilibrium. Free peptides were uniformly dispersed throughout thesolution volume while the relatively massive particles were depletedfrom the meniscus along with the peptides bound to them. Therefore, theamount of free vs SCK-bound peptide in solution was determined by theratio of absorbance at the meniscus to the absorbance of theunfunctionalized micelle and SCK. No absorbance for the tyrosine (λ₂₇₆)was detected near the meniscus in the 0.5% and 1.0% solutions relativeto controls at 5,000 rpm, indicating that all of the absorbance from thetyrosine residue of the PTD sequence is associated with the SCKnanoparticle as shown in FIG. 13(B).

A slight amount of absorbance observed for the 2.0% functionalized SCKsolution suggested free peptide in solution however, increasing therotor speed to 8,000 rpm, which depleted the meniscus of nanoparticles,indicated that no free peptide was present in the solution (FIG. 4).This measurement is of particular importance for two reasons: (1) thepresence of free peptide can compete with and inhibit the binding of thebioconjugate, and (2) the PTD sequence has measurable effects on invitro cell viability and inflammatory response.

FIG. 13(A) shows sedimentation equilibrium profiles collected using aninterferometry detector for the micelles, SCKs, andpeptide-functionalized nanoparticles. The dramatic increase in weightaverage molecular weight (M_(w)) was due to a small percentage ofinterparticle crosslinking (covalent or electrostatic), whichcontributed significantly to the molecular weight calculations, FIG.13(B) shows UV spectra, recorded near the meniscus of solutions atsedimentation equilibrium, showed negligible absorbance due to freetyrosine residues within the PTD, relative to controls. FIG. 13(C) showsrepeating the test at 8,000 rpm indicated that the meniscus was fullydepleted of SCK at 8,000 rpm, but not at 5,000 rpm, and also confirmedthat all the PTD was attached to the SCK.

Particle Analysis. The SCK nanoparticles were characterized extensivelyto probe for the differences in size, surface charge, and morphology asa result of post-cross-linking functionalization with the arginine-richPTD sequence. Dynamic light scattering (DLS) measurements were collectedin PBS, pH 7.4. This precaution was used to minimize dilute solutionaggregation of the nanoparticles. The polymeric micelle precursor to theSCK was shown to have a number-average diameter, D_(n), of 38±3 nm. Thecorresponding SCK, prepared with 50% cross-linking of its shell, wasfound to have a D_(n) of 37±2 nm. A comparison of D_(n) valuescalculated using the nonnegatively constrained least squares (NNLS)model for the diameter distributions of the 0.5% (7), 1.0% (8), and 2.0%(9), PTD-functionalized nanoparticles resulted in values of 35±3, 36±3,and 32±4 nm, respectively. When the volume-average diameter, D_(v), andthe intensity-average diameter, D_(z), distributions for thenanoparticles were calculated, the presence of small amounts ofaggregated species, representing a maximum of 5 vol %, was apparent inthe D_(v) and D_(z) diameter distributions. The likely source of theselarger diameter fractions is interparticle crosslinking due to thepresence of the PTD sequence; however, it is unknown whether the natureof the aggregation arises from covalent cross-linking or electrostaticattraction. These findings are consistent with the large increases notedin M_(w) for the PTD-functionalized SCKs relative to M_(w) obtained forpreformed SCK, 6. Since these aggregated species were less than 100 nmin diameter, filtration and/or dialysis were not effective in theirremoval from the majority population of smaller nanoparticles. However,because these species represent less than 1% of the nanoparticlepopulation by number, the contribution of these aggregates to data setscollected using imaging methods, such as atomic force microscopy (AFM),transmission electron microscopy (TEM) and confocal fluorescencemicroscopy, was negligible. Analysis of the SCKs by TEM depictedcircular shapes that possessed narrow size distributions. The observedTEM nanoparticle diameters were smaller than those measured as D_(n) inbuffer solution, due to drying and the effects of the phosphotunsticacid staining process employed during the TEM sample preparation. Zetapotential measurements also showed a diminishment in the negativesurface charge density with increasing functionalization. The zetapotential (ζ) data for the functionalized SCKs are included in Table 1.

Quantification of PTD Functionalization. The number of peptides perparticle in each sample was quantified using UV-visible spectroscopy andreaction of phenylglyoxal with the guanidine functionality of arginineas two independent methods of analysis. The number of peptides perparticle was measured from UV spectroscopy (Equations 1-9) by solvingsimultaneously for the peptide and SCK concentration using absorbancemeasurements recorded at two different wavelengths, λ₂₃₀ and λ₂₇₆.Analysis of SCK samples 7, 8, and 9 resulted in values of 38, 86, and˜500 peptides per particle, respectively. The large number of peptidesmeasured in sample 9 is a result of a slight amount of turbidity arisingfrom aggregation, which dominates the absorbance measurement at 230 nmas shown in FIG. 14. The coupling efficiency was approximately 80% andcorrelated well with the expected peptide numbers.

FIG. 14 shows UV-visible spectroscopy afforded the quantitativecalculation of the number of peptides per particle by two differentmethods. The first method involved the simultaneous solving of twounknowns as determined from PTD (A) and SCK (B) concentrationcalibration plots at λ₂₃₀ and λ₂₇₆. From the calibration curve generatedfrom FIG. 14(A) and FIG. 14(B), the concentration of PTD in therespective SCK solutions (FIG. 14(C)) was calculated. The large numberof peptides calculated to be in the 2.0% sample is a result of theslight amount of turbidity present in that sample due to aggregation,which is reflected in the absorbance measurement of that sample(orange). The second method involves the measurement of phenylglyoxalderivative formation (FIG. 14(D)). Phenylglyoxal reacts specificallywith the guanidine groups on the side chains of arginine residuesyielding a derivative with an absorbance red-shifted (λ₃₁₀) from that ofthe parent compound. From a calibration curve generated from knownamounts of PTD the molar concentration of the peptide in unknownsolutions was determined. Molar ratios were then applied to quantify thenumber of peptides per particle.

The second assay performed to quantify the number of peptides perparticle employed absorbance measurements at 310 nm, which exhibited anegligible contribution from solution turbidity. Phenylglyoxal reactsspecifically with the guanidine group of arginine residues under mildconditions. The PTD sequence contains six arginine residues which reactwith one phenylglyoxal moiety each in a mechanism outlined by Jairajpuriet al. (Biochem., 1998, 37, 10780-10791).

Measurements made using this method, again comparing molar ratios,resulted in degrees of functionalization of 41, 83, and 202 peptides for7, 8, and 9, respectively. These values are in good agreement with thedual wavelength, simultaneous UV spectroscopic determinations for thedegree of functionalization in 7 and 8. The value 202 determined by thephenylglyoxal method for 9 was in better agreement with expected valuesbased on the measured M_(w) for the SCK (Table 1). The validity of thephenylglyoxal method as a specific assay for the PTD moieties attachedto a SCK was also confirmed by performing the same testal conditions forthe nonfunctionalized SCK, as a negative control test, which showed noabsorbance due to the phenylglyoxal derivatization and thus no reactionof phenylygloxal with the unfunctionalized SCK, (6).

FIG. 15 shows live cell confocal fluorescence microscopy showingsuccessful internalization of the 0.5, 1.0 and 2.0% PTD-functionalizednanoparticles at both 37° C. and 4° C. in CHO cells. No uptake orinternalization was seen for the unfunctionalized parent SCK under livecell conditions.

Cell Transduction Tests. A mixture of 5 and (6)-carboxy-fluoresceinderivatized with a 2,2′-(ethylenedioxy)bis-(ethylamine) linker wasconjugated to the PTD-functionalized nanoparticles to visually imagebinding interactions of the conjugates with mammalian cells usingfluorescence microscopy. This type of fluorophore derivatization doesnot contribute to enhanced cellular uptake. The conjugation wasperformed on a molar equivalence basis and resulted in approximately 70dye molecules per particle, assuming complete conversion. Free dye didnot contribute to the analysis, as it was removed via dialysis againstPBS. The cell uptake of PTD-functionalized SCKs was investigated by thein vitro transduction of CHO cells. Numerous examples exist describingthe sequence specificity, mechanism, cofactor, and cell-type dependenceon the efficiency of cellular uptake. The mechanism, efficiency, andenergy dependence of the particle translocation was investigated in thisinstance by varying the metabolic conditions of the tests. Conditionsincluded 1 h incubation times at 37° C. and 4° C. in serum free media,and in the presence of 0.1% sodium azide. Bright field, fluorescence,and confocal microscopies were performed on live cells as well asspecimens that were fixed through 1 h incubations at ambient temperaturein a 4% paraformaldehyde solution. Confocal microscopy of fixed cellsclearly showed an accumulation of the fluorescent PTD functionalizednanoparticles inside the cells in all cases. Although thenonfunctionalized nanoparticles are also internalized, the apparentfluorescence intensities are qualitatively less than those of the PTDfunctionalized particles. Since fixation of cells involves the inherentperturbation of the cellular membrane, live cells were viewed withoutfixation to see whether rapid internalization occurred without thebenefit of fixation.

FIG. 16 shows confocal fluorescence microscopy of PTD-functionalizedSCKs using CHO cells fixed with a 4% paraformaldehyde solution commonlyused in the literature, shows nonspecific uptake of thenonfunctionalized SCKs. which was not observed in the live cell tests,in addition to the qualitatively enhanced uptake in each of the PTDfunctionalized samples. FIG. 16(A) and FIG. 16(B) show the confocalreconstructions of the respective SCK samples at 37° C. and 4° C.,respectively, following 1 h incubations and cell fixation. FIG. 16(C)shows CHO cells that were incubated for 1 h in 0.1% sodium azide withthe SCK samples.

In the cases where the cells were not fixed with the 4% paraformaldehydesolution, the non-PTD-functionalized SCKs were not observed in theintracellular space under fluorescence conditions when incubated at 37°C. or 4° C., and therefore it was determined that they were either notinternalized nonspecifically or not at a level that was readilymeasurable using optical or fluorescence microscopies. However, the0.5%, 1.0%, and 2.0% PTD-functionalized SCK nano-particles were readilyinternalized at 37° C., 4° C., and in the presence of 0.1% sodium azide,regardless of disruptive fixative processes that may or may notcontribute to an enhanced nonspecific effect.

Flow Cytometry Analysis. The efficiency of PTD-functionalized SCK celltransduction was quantified by fluorescence activated cell sorting(FACS). Data in Table 2 following, which indicate the percentage ofcells that are fluorescent for the respective samples, depict a trend ofincreasing cell transduction efficiency with increasing PTD/SCKstoichiometry. The slight decrease in the efficiency of uptake at 4° C.and in the presence of sodium azide, which depletes cellular ATP,relative to measured values at physiological temperatures show thatendocytotic processes are also contributing to the SCK internalization.However, it is evident that non-endocytotic pathways contributesignificantly to cell entry. While FACS analysis of thePTD-functionalized SCKs suggests multiple mechanisms of uptake, theintracellular internalization as visualized by live cell confocalmicroscopy is evident

FACS analysis does not discriminate between internalized and surfacebound particles, and efforts to further quantify the efficiency ofuptake and exchange of the respective samples by radiolabeling methodsare ongoing. The involvement of a single cellular process governing theinternalization process has been shown to be increasing unlikely. Whilethe ionic interactions between the TAT-derivatized species and the cellmembrane are generally thought to be the initial step in theinternalization process, the overall negative surface charge density, asmeasured by zeta potential, of the PTD-functionalized SCK nanoparticlessuggest that further evidence is required to bolster that assessment.Recently, Dowdy et al. (Nat. Med., 2004, 10, 310-315) have shown thatTAT-fusion proteins were internalized in a multistep process startingwith a receptor-independent association of the fusion protein with thecell surface followed by a lipid raft-dependent macropinocytosis, whichwas demonstrated to be independent of interleukin-2 receptor/raft-,caveolar- and clathrin-mediated endocytosis. While these findings shedcritical new and thorough insights on the mechanism of internalization,additional research on the nature of the initial construct-cell surfaceassociation step is needed.

The assembly of well-defined polymeric nanoparticles has been described,and subsequent functionalization of the SCK nanoparticles with PTD hasbeen shown to facilitate their transduction across cellular membranes.The numbers of peptides per SCK were controlled through stoichiometricbalance and measured by two independent methods. The feasibility andefficiency of intracellular internalization were quantified andconfirmed. TABLE 1 Summary of Physical Characterization Data Collectedfor the Parent SCK Nanoparticle and the PTD-Functionalized SCKs no. ofDLS D_(n) particle ν(L/g) M_(w) (×10⁶ Da) peptides ζ(V) (nm) TEM D_(av)(nm) micelle n.d. n.d. 0 −27 ± 0.7 38 ± 3 21.3 ± 3.8 SCK 0.556 ± 0.0074.004 ± 0.151 0 −20 ± 0.5 37 ± 2 18.3 ± 3.6 0.5% 0.587 ± 0.009^(a) 5.900± 0.241 52 ± 2 −28 ± 0.9 35 ± 3 20.2 ± 3.7 1.0% 0.622 ± 0.009 7.017 ±0.304 104 ± 4  −23 ± 0.7 36 ± 3 21.4 ± 3.6 2.0% 0.661 ± 0.007 7.818 ±0.248 210 ± 10 −22 ± 0.5 32 ± 4 21.6 ± 3.3

TABLE 2 the Quantification of PTD Functionalization in Each of theRespective SCK Samples Using Two Different Methods Afforded SimilarNumbers of Peptides Per Particle no. of no. of no. of peptides particlepeptides nominal peptides UV-vis phenylglyoxal SCK 0 0 0 0.5% 52 ± 2 3841 1.0% 104 ± 4  87 83 2.0% 210 ± 10 ˜500 202

TABLE 3 Percentage of Fluorescent CHO Cells Following the Transductionof Fluorescein-Labeled SCKs and PTD-Functionalized SCKs Quantified byFluorescence-Activated Cell Sorting particle 37° C. 4° C. 37° C. w/NaN₃4° C. w/NaN₃ SCK 5.6 7.3 5.2 7.1 0.5% 33.9 10.3 16.3 8.5 1.0% 42.9 32.625.7 12.3 2.0% 47.0 36.4 51.8 13.8

EXAMPLE SET C

Peptide-Derivatized Shell-Cross-Linked Nanoparticles and AssociatedBiocompatibility Evaluation

The inventors used conjugation of the protein transduction domain (PTD)from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles asa method to successfully facilitate cell surface binding and celltransduction.

Following assembly, the constructs (SCKs) were evaluated in vitro and invivo to obtain a preliminary biocompatibility assessment. The effects ofSCK exposure on cell viability were evaluated using a metabolic3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)and a fluorescent apoptosis assay. Furthermore, stages of apoptosis werequantified by flow cytometry. Although higher levels of peptidefunctionalization resulted in decreased metabolic function as measuredby MTT assay, advantageously significant apoptosis was not observedbelow 500 mg/L for our samples.

To evaluate the potential immunogenic response of thepeptide-derivatized constructs, a real-time polymerase chain reaction(RT-PCR) system that allows for the in vitro analysis and quantificationof the cellular inflammatory responses tumor necrosis factor alpha(TNF-α) and interleukin-1 beta (IL1-β) was utilized. The inflammatoryresponse to the peptide-functionalized SCK nanoparticles as measured byRT-PCR show statistically significant increases in the levels of bothTNF-α and IL1-β relative to tissue culture polystyrene (TCPS).Fortunately, the measured cytokine levels did not preclude the furthertesting of SCKs in an in vivo mouse immunization protocol. In thislimited assay living mice, measured increases in immunoglobulin G (IgG)concentration in the sera were minimal with no specific interactionsbeing isolated, and more importantly, none of the mice (>50) subjectedto the three 100 μg immunization protocol have died. Additionally,advantageously no gross morphological changes were observed inpostmortem organ histology examinations.

Test Procedures

Materials. Fmoc-protected amino acids and preloaded solid-phase Wangresins were purchased from NovaBiochem-CalBiochem Corp (San Diego,Calif.). Sodium dodecyl sulfate (SDS),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (98%)(MTT), Tween20, mouse IgG (Fc, Sigma I-8765), and goat anti-mouse IgGalkaline phosphatase conjugate (Fc specific, Sigma A-1418) werepurchased from Sigma Aldrick (St. Louis, Mo.). Alkaline phosphatesubstrate solution was purchased from BioRad (Hercules, Calif.).Spectra/Por dialysis membranes (Spectrum Laboratories, Inc., RanchoDominguez, Calif.), 96-well polystyrene plates (Falcon), and ELISAplates (Nunc-Immuno, Maxisorp) were purchased from Fisher ScientificCompany (Pittsburgh, Pa.). QuantiTect SYBR Green RT-PCR Kit and RneasyKit were obtained from Qiagen (Valencia, Calif.). Primer identification,isolation, and probe development were nearly identical to methodsdescribed in Bailey et al. (J. Biomed. Mater. Res., 2004, 69, 305-313and J. Dental. Res., 2004).

Certain commercial materials and equipment are identified in thisspecification in order to specify adequately the test procedure. In noinstance does such identification imply recommendation by the NationalInstitute of Standards and Technology nor does it imply that thematerial or equipment identified is necessarily the best available forthis purpose.

The Animal Facility at the Washington University Department of Biologyin St. Louis, St. Louis, Mo. performed immunizations, sera collections,and organ histology. Animal studies were conducted under Protocol20010343 approved by the Animal Studies Committee of WashingtonUniversity in St. Louis, St. Louis, Mo.

Cell Lines. Mouse myeloma B cells (ATCC, Manassas, Va.), CRL-1581.1, andCHO cells (ATCC) were obtained as gifts from Washington University inSt. Louis, St. Louis, Mo. Mouse myeloma B cells were cultured in RPMI(Life Technologies, Rockville, Md., USA) 1640 medium using TCM serumreplacement (10%, volume fraction) obtained from Celox Laboratories (St.Paul, Minn.). RAW 264.7 cells were purchased from ATCC. RAW 264.7 andCHO cells were maintained in RPMI supplemented with 10% (volumefraction) heat-inactivated fetal bovine serum (FBS, Life Technologies,Rockville, Md., USA), in 5% CO₂:95% air (volume fractions) at 37° C. Toharvest, RAW 264.7 cells were washed with calcium- and magnesium-freephosphate-buffered saline and subsequently incubated with Hank balancedsalt solution (HBSS) to promote release from the flask.

Determining Cell Viability via MTT Assay. Mouse myeloma B cells werecounted and diluted into fresh media at concentrations of 50,000cells/mL and an aliquot (100 μL) was seeded to each well in a 96-wellplate. After deposition of the cell suspension, the plates were placedin an incubator (37° C., 5% CO₂, volume fraction) and allowed to growfor 24 h. Fresh 96-well plates were loaded with sterile buffer (40 μL),and the first column of each row was loaded with polymer or SCK stocksolutions of known concentration. The first columns in each plate werethen diluted serially from columns 2 to 11. The last columns were heldas controls with no polymer or SCK added. Fresh medium (50 μL) was thenadded to every well in the plates. The original plates were removed fromthe incubator, and the premixed sample solutions were added to thecorresponding wells in each plate. After 24 h incubation, the inoculatedplates were removed from the incubator, and an aliquot of MTT (Sigma)(20 μL, 5.00 mg/mL) in PBS (0.05 mol/L phosphate, 0.05 mol/L sodiumchloride, pH 7.4) was added to each well. The plates were returned tothe incubator and allowed to equilibrate for 2 h. After 2 h, extractionbuffer (80 mL, 20% (mass fraction) SDS (Sigma) in 50:50 DMF:H₂O, volumefraction, pH 4.7) was added to each well to extract the aqueousinsoluble formazan product. The plates were then returned to theincubator for 18 h to allow for the extraction after which theabsorbance of each solution was measured at 560 nm. Concentrations weredetermined by the weight of lyophilized polymer sample dissolved in PBSand then adjusted for the dilution of the medium. Results are an averageof four values, and the standard deviation is reported for eachconcentration.

Apoptosis Assay. Apoptotic analysis of RAW 264.7 cells was assessedusing the Guava Nexin Kit (Guava Technologies, Hayward, Calif.). RAW264.7 cells were plated in 12-well plates at 50,000 to 100,000 cells perwell and allowed to adhere for 24 h, after which the cells wereinoculated with 100 μL aliquots of the respective samples. The initialquantity of SCK was suspended in 0.500 mL of PBS. The respective sampleswere subsequently added to RAW 264.7 cultures (100 μL). After 24 h, themedia was removed from the cells, and the cultures were washed with PBS.To promote release from the plate, the cells were incubated with 1 mL ofHBSS for 30 min. The cells were then gently removed from the plate, andthe volume was adjusted to 300 μL. The cell suspension was incubatedwith 5 μL of Nexin 7-AAD and 5 μL of Annexin V-PE for 5 min in alight-protected environment and subsequently analyzed.

Flow Cytometry. Apoptotic analysis of RAW 264.7 cells incubated on thethin films or in the presence of the nanoparticles was assessed usingthe Guava Nexin Kit (Guava Technologies, Hayward, Calif.). RAW 264.7cells were plated in 24-well plates (50,000 to 100,000) cells per welland allowed to adhere on tyrosine-derived polycarbonate thin films ortissue culture polystyrene for 24 h prior to analysis by flowcytommetry. Full testal details were described previously.

mRNA Extraction. Cells were plated in sterile 150 mm×25 mm nonpyrogenicpolystyrene dishes (Daigger, Vernon Hills, Ill.). SCK nanoparticles wereadded to plated cells 24 h following seeding. The mRNA extraction wascarried out using the materials and protocol provided in the Rneasy Kitfrom Qiagen (Valencia, Calif.). The mRNA extraction protocol wasfollowed according to the manufacturer's specification, except a21-gauge needle was used to homogenize the sample. The RNA was treatedwith RNA Secure immediately following elution and stored at −20° C.Standard spectrophotometric measurements were taken, and a 2% (massfraction) agarose gel stained with 10 μg/mL ethidium bromide (Sigma, St.Louis, Mo.) was used to image the RNA. Densitometry was performed usingthe Versa Doc imaging system (Bio-Rad, Hercules, Calif.).

Standards. The plasmids containing the cDNA inserts for TNF-α, IL-1β,and the 18 S ribosomal subunit were purchased from ATCC. The plasmidswere grown in luria-bertani (LB) medium (ATCC, medium 1065) with 100μg/mL of ampicillin for selection purposes. Plasmid DNA was isolatedusing the Plasmid Giga Kit (Qiagen, Valencia, Calif.) following theQiagen's protocol. Spectrophotometric measurements were made at 260 nm,and a 1% (mass fraction) agarose gel stained with 10 μg/mL ethidiumbromide (Sigma, St. Louis, Mo.) was used to image the DNA. Densitometrywas performed using the Versa Doc imaging system (Bio-Rad, Hercules,Calif.).

Primer Design. Primers were designed using Primerfinder (WhiteheadInstitute for Biomedical Research) for the RT-PCR tests. The primersgenerated were used in both PCR and RT-PCR tests. They are as follows:18S: 5′ AGCGACCAAAGGAACCATAA 3′ and 3′ CTCCTCCTCCTCCTCTCTCG 5′; TNF-α:5′ TTTCCTCCCAATACCCCTTC 3′ and 3′ AGTGCAAAGGCTCCAAAGAA 5′; IL-1β: 5′TGTGAAATGCCACCTTTTGA 3′ and 3′ GTAGCTGCCACAGCTTCTCC 5′.

The amplicons generated from these primers are 204 base pairs, 202 basepairs, and 205 base pairs, respectively. DNA sequencing was performedusing the Big Dye Terminator Kit (ABI, Foster City, Calif.) on a 310 DNAGenetic Analyzer (ABI, Foster City, Calif.).

RT-PCR. RT-PCR was carried out using the QuantiTect SYBR Green RT-PCRKit and protocol (Qiagen, Valencia, Calif.). All RT-PCR tests wereperformed using the iCycler (Bio-Rad, Hercules, Calif.). The protocolutilizes the following thermal parameters: Reverse Transcription: 30 minat 50° C. Activation step: 15 min at 95° C. Three-step cycling:denaturation for 30 seconds at 95° C., annealing for 2 min at 57° C.,extension for 2 min at 72° C. for 45 cycles. A melt curve wassubsequently performed to analyze the products generated, which began at50° C. and increased to 95° C. in 1° C. increments.

Immunization of Mice. For each sample, five live mice (female, balb c, 7weeks old) were inoculated with 100 μL of polymer or SCK solution (1mg/mL in PBS) at 0, 4, and 8 weeks. The mice were bled forpreimmunoserum screening prior to the primary injection. Blood sampleswere then collected 14 d after 4 and 8-week booster immunizations,respectively. Serum was obtained after clotting at 4° C., for 24 h, andcentrifugation. Serum samples were stored at −20° C. in small aliquotsfor later use.

Determining Antibody Titer by ELISA Assay. Samples for ELISA for thefive mice were first prepared by dilution in five 96-well plates. PBSbuffer (100 μL) was added to each well in the plates. The serum vialswere thawed, and each serum sample (5 μL) was diluted 1:100 in PBS andwas transferred, in quadruplicate, in aliquots (100 μL) to the firstfour wells in the top row of each corresponding plate. The serumobtained following the first booster immunization was added to the nextfour wells, and the serum obtained following the second boosterimmunization was added to the final four wells, columns 9 to 12. Theserum was then serially diluted 2-fold in rows 2-7 in each plate.Following dilution, 100 μL from each well was transferred to acorresponding ELISA (Nunc-Immuno, Maxisorp) plate and incubatedovernight at 4° C. The wells were washed three times with PBS (300 μL)containing 0.05% (mass fraction) Tween 20 (Sigma), and the remainingbinding sites on the plates were blocked with sodium caseinate (100 μL,2.5%, mass fraction, in PBS) followed by overnight incubation at 4° C.The wells were again washed three times, and a 1:40,000 dilution of goatanti-mouse IgG alkaline phosphatase conjugate (100 μL, Sigma) was addedto each well and allowed to incubate for 1 h at room temperature beforebeing washed five times. Alkaline phosphatase substrate solution (100μL, Bio-Rad) was added to each well. Color development was allowed totake place for 30 min and then was quenched with 4 mol/L H₂SO₄ (50 μLper well). Absorption measurements were made at 405 nm. To determine theabsolute antibody concentrations, mouse IgG standards were run inparallel with each ELISA plate. Serial dilutions of mouse IgG (from 5.00to 0.08 μg/mL) were used to generate the linear calibration plot ofabsorbance vs concentration. The value corresponding to 0 g/mL was thebackground reading and was subtracted from all samples.

Results and Discussion

The assessment of material biocompatibility is a complicated process,which includes both in vitro and in vivo measurement methods, each ofwhich depend on the physical and chemical nature of the material and thenature of the biological interaction. Surface characteristics such ashydrophobicity, morphology, surface charge, and chemical functionalityare all known to play key roles in governing cell adhesion andproliferation. Several physicochemical parameters for thePTD-derivatized SCK nanoparticles whose syntheses and characterizationare described in the previous example (B) are outlined in Table 1.However, a clear framework outlining the critical physiochemical andsurface-mediated interactions within which to facilitate the developmentof materials minimally detrimental to cells does not exist. Forapplications concerning the delivery of macromolecules into cells, theenhanced efficiency of conjugate transduction must be weighed againstany detrimental biological interactions including effects on both cellviability and immune response. The cell viabilities in the presence ofthe peptide-derivatized polymeric constructs were evaluated by MTTassay, which monitors enzymatic activity of a cell organelle (in thiscase mitochondria), and flow cytometry. Previously, the proteintransduction sequence has been used at concentrations of up to 100μmol/L without affecting the viability of certain cell lines (Suzuki etal., J. Biol. Chem., 2002, 277, 2437-2443).

As illustrated by the data shown in FIG. 7, the PTD sequence, with fouradditional glycine residues, was found to decrease the enzymaticreduction of MTT in the mitochondria of mouse myeloma B cells atapproximately 110 μmol/L concentrations. In contrast, the viabilities ofthe cells upon exposure to buffered solutions of micelles or SCKs remain70 to 90% at concentrations above 100 mg/L. The small drop in viabilityis due primarily to the dilution of the media as shown by control testsin which buffer and no buffer were added to different cell populations.Exposure of the cells to the parent PAA₅₆-b-PMA₁₈₅ micelles, 5, and thecorresponding unfunctionalized SCK, 6, did not affect cell viability upto 1125 mg/L concentrations. However, the derivatization of thenanoparticle with increasing numbers of the PTD sequence has asignificant effect on the viability of the cell populations as measuredby the enzymatic function. The 2.0% PTD-functionalized SCK 9 is toxic tothe cells between (140 and 280) mg/L (7 μmol/L to 13 μmol/L in PTD). The0.5% and 1.0% PTD-functionalized SCKs (7 and 8) are toxic between (560and 1125) mg/L (14 μmol/L to 28 mmol/L and 7 μmol/L to 15 μmol/L in PTD,respectively).

In addition to the MTT enzymatic assay, the peptide-derivatizednanoparticles have been evaluated by a fluorescent flow cytometry testto determine the effects of incubation with SCKs upon the induction ofapoptosis. Apoptosis is characterized by numerous morphological changes,the first of which involves the translocation of phosphatidylserine (PS)from the inner to the outer surface of the cellular plasma membrane.Once exposed to the extracellular environment, PS sites are accessibleto Annexin V, a phospholipid binding protein with a high affinity forPS. For measurement purposes, Annexin V is conjugated to FITC and usedfor the identification of cells in the early stages of apoptosis viaflow cytometry. Because PS binding sites are also accessible when thecells are in a necrotic state, Annexin V is not an absolute marker ofapoptosis. Therefore, it is often used in conjunction with7-aminoactinomysin, (7-AAD) which binds to exposed nucleic acids, whenthe cell membrane integrity is compromised. Cells that are negative(unstained) for both Annexin V and 7-AAD have no indications ofapoptosis: PS translocation has not occurred, and the plasma membraneremains intact. Cells that are Annexin V (+) and 7-AAD (−), however, arein early apoptosis, as PS binding sites are exposed, but the plasmamembrane is still intact. Cells that are (+) for both Annexin V and7-AAD are either in the late apoptosis (irreversible) stage or are notviable.

The flow cytometry data shown in FIG. 8 indicate that the SCKs do notinduce apoptosis at the concentrations tested. The total inoculatedconcentration was 0.5 mg/mL (500 mg/L) for each SCK sample. In addition,a visual assessment of the effects of exposure to PTD-derivatized SCKsat the 5 day time point indicated that the nanoparticles did not affectthe continued growth of the cells nor did they induce significantmorphological changes (data not shown). These results are comparable tothe MTT assay results. Although the MTT assay depicts a shutdown inmitochondrial function at certain concentrations, exposure of each ofthe samples to the cells at concentrations of 500 mg/L did not lead tocellular apoptosis.

Immunogenicity Results. The extent of PTD functionality is ofsignificant importance as it may affect the immune response. The natureof the response has significant clinical implications, as severeinflammatory responses may prevent in vivo applications. Recentliterature has reported that the addition of HIV-1 TAT PTD enhancesminigene epitope presentation in tissue culture, but the attachment ofPTD to full-length proteins does not enhance the immune response of theconstruct (Liefert et al., Gene Ther., 2002, 9, 1422-1428). The reporteddata were for a 1:1 stoichiometry of PTD:protein, and it is unclearwhether the presentation of multiple copies of the sequence on thesurface of an SCK would have a synergistic or negligible effect on thecellular immune response. The initiation and propagation of an immuneresponse is ultimately regulated by cytokines. Cytokines are the primarymediators of a host immune response and are able to both induceapoptosis and modulate survival through the activation of genes inresponse to bacterial toxins, inflammatory products, and other invasivestimuli. Two of the most important cytokines in the acute inflammatoryresponse are tumor necrosis factor alpha (TNF-α) and interleukin-1 beta(IL1-β). TNF-α is synthesized and secreted by macrophages. TNF-α is partof a complex network of cytokines and is capable of initiating cascades,which control the synthesis and expression of signaling molecules,hormones, and their receptors. Knowledge of the cellular cytokineprofile is key to identifying the processes involved in the immuneresponse and crucial to the further elucidation on the physicochemicalproperties of the bioconjugate that are inducing the response. Anotherprominent cytokine involved in the inflammatory responses isinterleukin-1 (IL-1). Although IL-1 protects the organism by enhancingthe response to pathogens and initiating healing process, itsoverproduction can induce numerous pathological consequences includingseptic shock and leukemia. The measurement of mRNA levels has been usedwidely to measure cytokine proliferation, which is responsible for theinitiation, mediation, and propagation of cellular inflammatoryresponses. The genetic expression profiles of IL-1β and TNF-α have beenmeasured by real-time polymerase chain reaction (RT-PCR). In this study,the mRNA profiles of TNF-α and IL-1β in response to PTD, SCKs, and SCKsderivatized with increasing amounts of PTD sequences were investigatedusing RT-PCR. In the previous example, PTD was conjugated in (0.005,0.01, and 0.02) molar ratios, relative to the acrylic acid residues inthe shell, to the SCK nanoparticles resulting in SCK populationsnominally possessing 52, 104, and 210 (41, 83, and 202 as measured byphenylglyoxal) PTD peptides per particle, respectively. RAW 264.7 cellswere used because they retain the characteristics of primary culturedmacrophages in vitro, including the ability to release cytokines. Cellswere exposed to the respective SCK nanoparticle samples by adding theconjugates to the murine macrophages 24 h following seeding. After a 24h incubation period, the mRNA was extracted from the cell populations.Using a reverse transcriptase enzyme, mRNA is converted to the cDNAtemplate necessary for amplification. Once cDNA is generated, genespecific primers, a DNA polymerase, and a fluorescent moiety areutilized to amplify and label the amplicon generated. The gene productaccumulation was then measured during the exponential phase of theamplification reaction. The copy number from each of the samples wasobtained by extrapolating to a standard gene curve of knownconcentration and copy number to yield quantitative data. The assay alsoincludes the analysis of mRNA that does not change in relative abundance(18S) during the course of treatment to serve as an internal control.The genetic profiles of the inflammatory response to SCKs (1-3), PTD(4), PTD-functionalized SCK nanoparticles (5-10), and tissue culturepolystyrene (TCPS, 11) are shown in FIG. 9.

TNF-α mRNA synthesis was up-regulated 2-fold to 14-fold (depending onconcentration) in response to exposure of the parent SCK nanoparticle,6, over TCPS. It is interesting to note that the highest concentration,10.0 mg/L, of the parent SCK 6 actually caused less of an increase,2.2-fold, than lower concentrations, 0.1 and 1.0 mg/L, of SCK 6,13.5-fold and 13.2-fold, respectively. When looking at the effects ofparent SCK concentration on TNF-α production, the difference between 0.1and 1.0 mg/mL is statistically insignificant, while the 10.0 mg/mLsample measures 6.1-fold less than at the 1.0 mg/mL level.Interestingly, the down-regulation of TNF-α has been demonstratedpreviously in liposome vectors formulated with cationic lipids. Themeasured TNF-α levels for the PTD-functionalized SCKs are less than thelevels of the parent SCK in all cases (1.5-fold to 3.0-fold) and show noconcentration or functionality dependence. The levels of TNF-measured inresponse to the peptide-functionalized nanoparticles, regardless of theextent of derivatization and concentration tested, are statisticallyinsignificant when compared to the levels induced by the PTD domainitself. The copy numbers, fold increases, and the p values (p<0.05significant at 95% confidence, derived from the students t-test)indicating statistical significance are listed in Table 2 following.

The measured increases of IL1-β production in response to PTD-, SCK-,and PTD-functionalized SCKs are much more pronounced than the copynumber levels of TNF-α as compared to TCPS. The measured levels rangefrom 18.8-fold to 483.5-fold increases over TCPS and 1-fold to 25.4-foldincrease over SCKs of similar concentration and show statisticaldependence with regard to both concentration and PTD functionalization.The differences in TNF-α and IL1-β expression measured in this seriesreflect different pathways of signal transduction with regard to boththe time frame and the severity of the response to the respectiveinflammatory stimuli.

Following the in vitro measurements on cell viability and inflammatoryresponse, the SCKs and PTD-derivatized SCKs were then tested in vivo togauge their immunogenic response in balb/c mice. Enzyme-linkedimmunosorbent assays (ELISA) were used to quantify the increases in IgGproduction in response to immunization with the test compounds. Effortsto isolate specific responses by plating the SCKs on the ELISA plateprior to serum incubation were not successful due to an insignificantspecific antibody response. This result can be attributed to twopossible factors: (1) the nanoparticles could induce a nonspecificimmune response, and (2) the sera used for the ELISA measurements werecollected 14 days following the respective immunization, a sufficientlylong time that any acute inflammatory response may have passed. Wetherefore decided to look at the increases in IgG concentration on anindividual mouse basis to look for relatively large increases. FIG. 9shows the three antibody titers determined for each mouse in the study.The PTD peptide by itself is not immunogenic after two boosterimmunizations, and the parent SCKs possessing poly(acrylicacid-co-acrylamide) shells display negligible values of titer increasesover the control PBS injections. Those SCKs possessing various amountsof PTD functionality, 0.5%, 1.0%, and 2.0% (7, 8, and 9), qualitativelyshowed a general trend of increasing antibody titer in the serum withincreasing degrees of nanoparticle functionalization. The relativeincreases in antibody titer relative to the initial value are listed inTable 2. Qualitatively, the only significant immunogenic responsedepicted arose from the 2.0% functionalized SCK, 9. The 0.5%, 1.0%, and2.0% SCKs (7, 8, and 9) exhibited increases of 100% or more in 20% (1 of5), 60% (3 of 5), and 100% (5 of 5) of the mice, respectively, followingtwo booster injections.

While limited in scope, the immunogenicity results for the SCKsfunctionalized to a lesser degree are consistent with the literature,but when large numbers of PTD are presented, the particles do elicit anincrease in IgG concentration within the serum. While no specificresponses could be detected for the any of the SCK nanoparticles, it isperhaps more important that no deaths resulted from the mice (>50) beingsubjected to the immunization protocol (3×100 μg) and no grossmorphological changes were observed in postmortem organ histologyexaminations.

SUMMARY

SCK nanoparticles were derivatized with various stoichiometricderivatizations with PTD and were evaluated in vitro and in vivo forbiocompatibility. The initial evaluation and early identification ofdetrimental interactions between biological species and SCKnanoparticles are crucial to further efforts to develop in vivoapplications. Apoptosis assays measured by flow cytometry havecomplemented enzymatic (MTT) toxicity results demonstrating a lack ofdetrimental effects on cell viability below 500 mg/L for all thesamples. Although higher levels of peptide functionalization resulted indecreased metabolic function, loss of cell viability was observed at asufficiently high concentration, suggesting the use of even 2%PTD-functionalized SCKs would not be prohibited in vivo. In addition,RT-PCR data provided quantitative information regarding the lack ofimmunogenicity elicited by SCKs and peptide-derivatized SCKnanoparticles. The inflammatory response to the peptide-functionalizedSCK nanoparticles as measured by RT-PCR show increases in the levels ofboth TNF-α and IL1-β relative to TCPS. Although these levels arestatistically significant and show large increases of IL1-β withincreasing peptide functionalization, the levels did not preclude thepreliminary testing of SCKs in vivo. An in vivo mouse immunization modelfound measured increases in IgG concentration were minimal with nospecific interactions being identified, and more importantly, none ofthe mice (>50) subjected to the three 100 μg immunization protocol died.Additionally, no gross morphological changes were observed in postmortemorgan histology examinations. While these in vitro assessments andpreliminary in vivo tests show promising results, additional in vivoanimal studies, which are currently in progress includingbiodistribution and nanoparticle clearance, are needed to complimentthese initial findings and to extend the potential therapeuticapplications of functionalized SCKs. However, up-regulation of IL1-β andlarger than expected IgG increases for the 2.0% PTD-functionalized SCKssuggest that nanoparticles functionalized in polyvalent strategies maybe interesting scaffolds for the attachment of known antigens for use invaccination applications. TABLE 1 Summary of Physical CharacterizationData Collected for the Parent SCK Nanoparticle and thePTD-Functionalized SCKs particle ν(L/g) M_(w) (×10⁶ Da) no. of peptidesζ(V) DLS D_(n) (nm) TEM D_(av) (nm) micelle n.d. n.d. 0 −27 ± 0.7 38 ± 321.3 ± 3.8 SCK 0.556 ± 0.007 4.004 ± 0.151 0 −20 ± 0.5 37 ± 2 18.3 ± 3.60.5% 0.587 ± 0.009^(a) 5.900 ± 0.241 52 ± 2 −28 ± 0.9 35 ± 3 20.2 ± 3.71.0% 0.622 ± 0.009 7.017 ± 0.304 104 ± 4  −23 ± 0.7 36 ± 3 21.4 ± 3.62.0% 0.661 ± 0.007 7.818 ± 0.248 210 ± 10 −22 ± 0.5 32 ± 4 21.6 ± 3.3

TABLE 2 the Quantification of PTD Functionalization in Each of theRespective SCK Samples Using Two Different Methods Afforded SimilarNumbers of Peptides Per Particle no. of no. of no. of peptides particlepeptides nominal peptides UV-vis phenylglyoxal SCK 0 0 0 0.5% 52 ± 2 3841 1.0% 104 ± 4  87 83 2.0% 210 ± 10 ˜500 202

TABLE 3 Relative Percent Increase in Antibody Titers Quantified by ELISAFollowing Two Booster Injections over Preimmunization Baseline Levels.Particle 37° C. 4° C. 37° C. w/NaN₃ 4° C. w/NaN₃ SCK 5.6 7.3 5.2 7.10.5% 33.9 10.3 16.3 8.5 1.0% 42.9 32.6 25.7 12.3 2.0% 47.0 36.4 51.813.8

The present discovery provides novel systems and compositions for thetreatment and monitoring of diseases (e.g., cancer). For example, thepresent discovery provides systems and compositions that target, andsense pathophysiological defects, allow for imaging of the defects,provide the appropriate therapeutic based on the diseased state, monitorthe response to the delivered therapeutic, and identify residualdisease. In preferred embodiments of the present discovery, thecompositions are small enough to readily enter a patient's or subjectscells.

EXAMPLE—SET D

Micropet Imaging of MCF-7 Tumors in Mice Via unr mRNA-Targeted PNAs

In this example the inventors designed and synthesized three antisenseand one sense hybrid PNAs (peptide nucleic acids) with a four-lysinetail at the carboxy terminus to the unr mRNA that is highly andabundantly overexpressed in a breast cancer cell line (MCF-7). A DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) chelatingmoiety was added to the amino terminal end of the PNAs so that theycould be radiolabeled with ⁶⁴Cu for biodistribution and microPET imagingstudies in normal and MCF-7 tumor bearing mice. All four PNA conjugateswere successfully labeled with ⁶⁴Cu under mild conditions with aspecific activity of ca. 200 mCi/μmol (or 7,400 MBq/μmol) at time ofinjection. Biodistribution of two ⁶⁴Cu-labeled conjugates with antisenseand sense sequences of unr mRNA (PNA50 and PNA50S) were performed innormal balb/c mice via two injection modes (intravenous andintraperitoneal). Both conjugates exhibited a rather similar in vivobehavior featuring high uptake and long retention in kidney, and lowuptake and efficient clearance in blood and muscle. The administrationforms did not change the pattern of tissue distribution, while theintraperitoneal mode gave a much slower release rate of the conjugates.The MCF-7 tumor (100-320 mg) in CB-17 severe combined immunodeficiency(SCID) mice was imaged with all four ⁶⁴Cu-labeled PNA conjugates bymicroPET probably because of the high specific activity, but the imagecontrast varies with different time points and different compounds. Thequantification of microPET images was carried out to evaluate theconcentration of the radiolabeled PNA conjugates in the regions ofinterest (ROIs), and the results are in agreement with the data of postimaging biodistribution study (24 h post injection). Of the PNAconjugates studied in this work, ⁶⁴Cu-DOTA-Y-PNA50-K4 showed the bestimage quality of the tumor at all time points. As determined by the postimaging biodistribution, the tumor/muscle ratio (6.6±1.1) of⁶⁴Cu-DOTA-Y-PNA50-K4 is among the highest reported for radiolabeledoligonucleotides. Our work further strengthens the potential of antigeneand antisense PNAs to be utilized as specific molecular probes for earlydetection of cancer and ultimately for patient-specific radiotherapy.

Test Procedures

Materials. DOTA-tris(t-Butyl ester) was purchased from Macrocyclics Inc.(Dallas, Tex.), diisopropylethylamine (DIEA), TFA, m-cresol and diethylether (anhydride) were purchased from Aldrich (St. Louis, Mo.).Fmoc-protected amino acids (D-Lys(Boc)-OH and Tyr(tBu)-OH) werepurchased from NovaBiochem (La Jolla, Calif.).O-(7-Azabenzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), Fmoc-XAL PEG-PS resin, PNA building blocks(Fmoc-A-(Bhoc)-OH, Fmoc-C-(Bhoc)-OH, Fmoc-G-(Bhoc)-OH and Fmoc-T-OH) andother reagents and solvents for PNA and peptide synthesis were purchasedfrom Applied Biosystems (Foster City, Calif.). UV spectral data wereacquired on a Bausch and Lomb Spectronic 1001 spectrophotometer orVarian Cary 100 Bio UV-Visible Spectrophotometer. Matrix-assisted laserdesorption ionization (MALDI) mass spectra of PNA-peptide conjugateswere measured on PerSeptive Voyager RP MALDI-time of flight (TOF) massspectrometer using sinapinic acid as a matrix and calibrated versusinsulin (average [M+H⁺]=5734.5) that was present as an internalstandard. High-pressure liquid chromatography was carried out on BeckmanCoulter System Gold 126 with an array detector. Copper-64 was preparedon the Washington University Medical School in St. Louis, Mo. CyclotronCS-15 cyclotron by the ⁶⁴Ni(p,n)⁶⁴ Cu nuclear reaction at a specificactivity of 50-200 mCi/μg at the end of bombardment (EOB) as previouslydescribed. Water was distilled and then deionized (18 MΩ/cm²) by passingthrough a Milli-Q water filtration system (Millipore Corp., Bedford,Mass.). Diethylenetriaminepentaacetic acid (DTPA), ammonium acetate, andsodium chloride were purchased from Fluka Chemie AG (Buchs,Switzerland). Dry powder in foil pouches for the preparation of 10 mMphosphate buffered saline (PBS), pH 7.4, and4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) werepurchased from Sigma-Aldrich (St. Louis, Mo.). Solvents (e.g., acetone,methanol, etc.) were purchased from Fisher Scientific (Pittsburgh, Pa.)and used as received. Saline (0.9% NaCl solution) was purchased fromAmerican Pharmaceutical Partners, Inc. (Schaumburg, Ill.). Centricons(YM-3: MWCO 3,000 Da) were purchased from Millipore Corporation(Bedford, Mass.). Fast protein liquid chromatography (FPLC) andradio-FPLC were performed using an Amersham Pharmacia Biotech ÄKTA FPLC(Amersham Biosciences Corp. Piscataway, N.J.) equipped with a Beckman170 Radioisotope Detector (Beckman Instruments, Inc. Fullerton, Calif.).The Superdex™ 75 was bought from Amersham Biosciences Corp. (Piscataway,N.J.). PBS, Trypsin/EDTA, and cell culture media and additives werepurchased from the tissue culture support center of WashingtonUniversity School of Medicine (St. Louis, Mo.). Fetal bovine serum(FBS), Earle's balanced salt solution (BSS), and insulin were boughtfrom Sigma (St. Louis, Mo.). CB-17 SCID mice were purchased from theCharles River Laboratories (Wilmington, Mass.).

Tissue culture and animal model. The MCF-7 cell line was obtained fromAmerican Type Culture Collection (ATCC, Manassas, Va.). MCF-7 cells weregrown in Eagle's minimum essential medium (MEM) with Earle's BSS and 2mM L-glutamine modified to contain 1 mM sodium pyruvate, 0.1 mMnon-essential amino acids, 1.5 mg/mL sodium bicarbonate supplementedwith 10% FBS and 0.01 mg/mL bovine insulin. To establish MCF-7 humanbreast xanografts, each CB-17 SCID mice was implanted with a 60-daysubcutaneous (s.c.) slow release estrogen pellets (1.7 mg17β-estradiol/pellet; Innovative Research of America, Sarasota, Fla.) 48h prior to the s.c. injection of MCF-7 cells into the nape of neck.Cultured MCF-7 cells were harvested from monolayer using PBS andtrypsin/EDTA, and suspended in media with FBS. The cell suspension wascentrifuged and resuspended in PBS at the concentration of 1×10⁸ cellsper milliliter. It was then mixed 1:1 with Matrigel and injected s.c.(1×10⁷ cells per mouse, injection volume 200 μL) into the nape of theneck of CB-17 SCID mice (5-6 weeks of age). After the cell injection,the animals were monitored twice a week by general observations. Thetumor was noticed to grow in the first week and allowed to grow fiveweeks to reach a palpable size for microPET imaging studies. The tumorweight was 100-320 mg as determined by post imaging biodistribution.

Synthesis of the DOTA-(D)-Y-PNA-K4 conjugates. NH₂-Y-PNA-K4 conjugateswere synthesized continuously on universal support XAL-PEG-PS resin on a2 μmol scale with standard solid phase Fmoc off chemistry in Expedite8909 Synthesizer (Applied Biosystems) by loading the fifth and sixthbuilding block channel with Fmoc-D-Lys and Fmoc Tyr(tBu) respectivelyand programming the sequence accordingly under standard automated PNAsynthesis protocol. DOTA-tris(t-Butyl ester) (11.5 mg, 20 μmol) wasdissolved in 100 μl NMP, then 100 μl HATU DMF solution (0.2 M) and 100μl base solution (0.2 M DIEA, 0.3 M 2,6-lutidine in DMF) were added, themixture was introduced into the cartridge containing PNA-K4-resinmanually with a syringe and pushed back and forth with a second syringeto agitate the resin suspension every 10 min for 1 hour. Then the resinwas washed with DMF and dichloromethane (DCM) and dried by passingnitrogen. Treatment of the resin with TFA/m-cresol (4:1) for 12 h atroom temperature (r.t.) was used to cleave the conjugates and remove theside chain protective groups. Ethyl ether (8-10 volumes) was added tothe TFA solution to precipitate the product as yellow solid. The crudeproduct was purified by reversed phase HPLC on a Microsorb C18 column(300 Å pore, 5 μm particle size, 4.6×250 mm) using a 5% to 70% lineargradient of solvent B (0.1% TFA in acetonitrile) in A (0.1% TFA inwater) over 65 min at the flow rate of 1 ml/min. The effluent wasmonitored by absorbance at 260 nm and the major peaks were collected,concentrated to dryness in vacuum, and characterized by MALDI-TOF massspectrometry.

Radiolabeling of DOTA-Y-PNA-K4 conjugates with ⁶⁴Cu. Copper-64 chloride(typically in 0.1 M HCl) was converted to Cu-citrate by adding anappropriate volume of 0.1M ammonium citrate buffer (pH 7.0) to the⁶⁴CuCl₂ solution. Prior to labeling, the stock solutions ofDOTA-Y-PNA-K4 conjugates were heated at 80° C. for 10 min to minimizethe self-pairing and plastic wall sticky property of the PNA conjugates.To 200 μL of a DOTA-Y-PNA-K4 conjugate solution (40 μM), 20 μL of⁶⁴Cu-citrate was added (2-4 mCi). The resulting solution was incubatedat 60° C. for 1-2 h in a thermomixer (1,000 rpm). After incubation, 5 μLof 10 mM DTPA solution was added to the ⁶⁴Cu-DOTA-Y-PNA-K4 solution. Thesolution was vortexed for a few seconds and left at r.t. for 10 min. Itwas then centrifuged at least two times through a Centricon-YM3 (MWCO3,000 Da) with 10 mM PBS buffer (pH 7.4) to remove the ⁶⁴Cu-DTPA complexand/or free ⁶⁴Cu-activity. The radiochemical purity (RCP) of the⁶⁴Cu-labeled DOTA-Y-PNA-K4 conjugate was monitored by FPLC. The productwas then diluted with 10 mM PBS buffer (pH 7.4) to prepare appropriatedoses for biodistribution and microPET imaging studies.

FPLC analysis. A 100 μL of analyte was injected into a Superdex™ 75 gelfiltration column, which was then eluted with 20 mM HEPES and 150 mMNaCl (pH 7.3) buffer at an isocratic flow rate of 0.5 mL/min. The UVwavelength was preset to 280 nm, the radioactivity was monitored by anonline Beckman radio-detector. Under these conditions, the retentiontimes of the ⁶⁴Cu-DOTA-Y-PNA-K4 and DOTA-Y-PNA-K4 were ca. 31-35 min.

Biodistribution studies. All animal studies were performed in compliancewith guidelines set by the Washington University in St. Louis, Mo.Animal Studies Committee. Copper-64 labeled DOTA-Y-PNA-K4 solutions werediluted with saline. Normal balb/c mice weighing 20-30 g (n=4 per timepoint) were anesthetized with isoflurane and injected with 10-12 μCi ofactivity via the tail vein (i.v.) or ca. 55 μCi of radioactivity viaintraperitoneal (i.p.) injection. The injected volume of activity permouse was 100 μL. The mice were anesthetized prior to sacrifice (bydecapitation) at each time point. Organs of interest were removed,weighed, and counted. Standards were prepared and counted along with thesamples to calculate the percent injected dose per gram (% ID/g) andpercent injected dose per organ (% ID/organ).

MicroPET imaging studies. The microPET imaging studies were carried outusing the microPET® R4 (rodent) scanner (Concorde Microsystems Inc.,Knoxville, Tenn.). MCF-7 tumor-bearing CB-17 SCID mice were anesthetizedwith 1-2% vaporized isoflurane and injected with ca. 200-400 μCi ofactivity in 120 μL saline via the tail vein (⁶⁴Cu-DOTA-Y-PNA50: 210 μCi;⁶⁴Cu-DOTA-Y-PNA50S: 347 μCi; ⁶⁴Cu-DOTA-Y-PNA5: 253 μCi;⁶⁴Cu-DOTA-Y-PNA7: 361 μCi). At specific time points (1 h, 4 h, and 24 h)post injection, the mice were re-anesthetized and then immobilized in asupine position on custom-built support beds with attached anestheticgas nose cones for data collection. Within 4 h p.i., the imagingcollection time was 10 min; at 24 h p.i., the imaging collection timewas 20 min.

Radioactive tracer accumulation (⁶⁴Cu-labed PNAs) in a targeted organwas measured using the standardized uptake value (SUV). The SUVs wereobtained by the quantification of the regions of interest (ROIs) byviewing these areas in the selected tissues and averaging the activityconcentration corrected for decay over the contained voxels (multipleimage slices) at the time points p.i.${SUV} = \frac{{Radioactivity}\quad{Concentration}\quad{in}\quad{{ROI}\left\lbrack {\mu\quad{Ci}\text{/}{cc}} \right\rbrack}}{{Injection}\quad{{{Dose}\left\lbrack {\mu\quad{Ci}} \right\rbrack}/{Animal}}\quad{{Weight}\lbrack g\rbrack}}$

After the microPET imaging at 24 h p.i., the animals were sacrificed andbiodistribution studies were performed. The ratios of tumor to blood(T/B) and tumor to muscle (T/M) were calculated. The unpaired t-test onthe biodistribution and microPET quantitation data was performed usingPrism, v. 4.00 (Graphpad, San Diego, Calif.).

Results

Selection of the antisense PNAs. The sequences of the antisense PNAswere selected by a procedure that will be described in greater detail inExample set E. Three of these antisense PNAs and one sense PNA wereselected for imaging studies: PNA50 with a K_(d) of 21 pM, PNA5, with aK_(d) of 22 pM, PNA7 with a K_(d) of 15 pM, and PNA50S (sense form ofPNA50) with a K_(d) of >10 nM in 0.1 M NaCl, 50 mM EDTA, 2 mM cacodylicacid.

Synthesis of DOTA-Y-PNA-K4 conjugates (Table 1). The PNAs weresynthesized using standard automated solid phase Fmoc synthesis on anABI 8909 DNA synthesizer with a PNA option (this synthesizer is nolonger commercially available from the original manufacturer). Theunnatural D-isomer of lysine was used to inhibit enzymatic degradationof the K4 permeation peptide unit. The DOTA group was added manually inthe last step of the synthesis to the amino terminal (“5′-end”) of thePNA via the commercially available tri-t-butylester, and the HPLCpurified products characterized by MALDI-TOF.

Radiolabeling of DOTA-Y-PNA-K4 conjugates. Four DOTA-Y-PNA-K4 conjugateswere all successfully radiolabeled with ⁶⁴Cu in 0.1 M ammonium citratebuffer (pH 7.0) under mild conditions (at 60° C. for 1-2 h) in yields of32-61% (decay corrected). After DTPA challenge of non-specifically bound⁶⁴Cu-activity and Centricon-YM3 (MWCO: 3,000 Da) separation, theradiochemical purity of the ⁶⁴Cu-labeled PNA conjugates was nearly 100%as determined by radio-FPLC: both the radioactivity and UV (280 nm)curves only showed a single strong peak with the same retention time inthe range of 30-35 min. The ⁶⁴Cu-labeled PNA conjugates remained 100%intact after being kept in saline overnight.

Biodistribution of ⁶⁴Cu-DOTA-Y-PNA-K4 conjugates in normal balb/c mice.In order to better evaluate the in vivo kinetics of the ⁶⁴Cu-labeled PNAconjugates, the biodistribution studies were carried out with twodifferent injection modes in normal balb/c mice: intravenous (tail vein)injection (i.v.) and intraperitoneal injection (i.p.).

Tail vein injection. The biodistribution data of ⁶⁴Cu-DOTA-Y-PNA50-K4and ⁶⁴Cu-DOTA-Y-PNA50S-K4 in blood, liver, kidneys, and muscle arepresented as percent of injected dose per organ (% ID/organ) at 20 min,1 h, and 4 h post injection (p.i.) in FIG. 32. Both conjugates exhibitedhigh kidney uptake and long retention. Within 1 h p.i.,⁶⁴Cu-DOTA-Y-PNA50-K4 showed slightly higher kidney accumulation(37.4±1.8% ID/kidney at 20 min p.i. and 45.8±0.7% ID/kidney at 1 h p.i.)than ⁶⁴Cu-DOTA-Y-PNA50S-K4 (35.6±0.6% ID/kidney at 20 min p.i. and42.3±0.2% ID/kidney at 1 h p.i.). Out to 4 h p.i., ⁶⁴Cu-DOTA-Y-PNA50-K4exhibited significant kidney clearance (36.1±3.6% ID/kidney at 4 h p.i.P <0.02 as compared to the value at 1 h p.i.), while a drasticaccumulation was observed for ⁶⁴Cu-DOTA-Y-PNA50S-K4 (60.5±3.6% ID/kidneyat 4 h p.i. P<0.005 as compared to the value at 1 h p.i.). In blood,liver, and muscle, the uptake of ⁶⁴Cu-DOTA-Y-PNA50-K4 was slightly lowerthan that of ⁶⁴Cu-DOTA-Y-PNA50S-K4 out to 4 h p.i. Both conjugatesshowed around 4% ID/organ of uptake in bone at 20 min p.i., but theywere cleared to <1% ID/organ at 4 h p.i. Negligible uptake was observedin lung, spleen, heart, and brain for both compounds (<1% ID/organ).

Intraperitoneal injection. The biodistribution of ⁶⁴Cu-DOTA-Y-PNA50-K4and ⁶⁴Cu-DOTA-Y-PNA50S-K4 via the intraperitoneal injection mode wasperformed in normal balb/c mice (n=3) at 24 h and 48 h p.i. Thebiodistribution data in selected organs are shown in FIG. 33. Bothcompounds exhibited similar uptake in kidney and liver at 24 h p.i.(⁶⁴Cu-DOTA-Y-PNA50-K4: 37.5±0.7% ID/kidney and 3.2±1.6% ID/liver;⁶⁴Cu-DOTA-Y-PNA50S-K4: 37.6±1.0% ID/kidney and 3.7±2.4% ID/liver), whichis comparable to the data of the i.v. mode at 20 min p.i. While nosignificant clearance was observed from kidney at 48 h p.i. for eithercompound (⁶⁴Cu-DOTA-Y-PNA50-K4: 33±6% ID/kidney; ⁶⁴Cu-DOTA-Y-PNA50S-K4:35±6% ID/kidney), it is apparent that both conjugates were accumulatingin liver out to 48 p.i. (⁶⁴Cu-DOTA-Y-PNA50-K4: 6.1±2.1% ID/liver;⁶⁴Cu-DOTA-Y-PNA50S-K4: 6.5±2.6% ID/liver). Surprisingly, the muscleuptake of ⁶⁴Cu-DOTA-Y-PNA50S-K4 drastically increased from 5.9±2.4%ID/muscle at 24 h p.i. to 25.0±4.4% ID/muscle at 48 h p.i., while⁶⁴Cu-DOTA-Y-PNA50-K4 maintained the same uptake level from 24 h(4.9±1.8% ID/muscle) to 48 h p.i. (4.5±0.2% ID/muscle). Both compoundsexhibited low accumulation in other tissues (<1% ID/organ in lung,spleen, heart, and brain; <2% ID/organ in blood and bone; and <3%ID/organ in fat at 48 h p.i.).

MicroPET imaging of ⁶⁴Cu-DOTA-Y-PNA-K4 conjugates. The microPET imagingwas performed in MCF-7 tumor-bearing CB-17 SCID mice with⁶⁴Cu-DOTA-Y-PNA-K4 conjugates at 1 h, 4 h, and 24 h p.i. Of the fourcompounds, ⁶⁴Cu-DOTA-Y-PNA50-K4 exhibited visually the highest imagecontrast of tumor, which was implanted in the nape of the neck, out to24 h p.i. (FIG. 34(a)). Consistent with the biodistribution results, themicroPET images (FIG. 35) showed that the kidneys were the primary organof accumulation of the four PNA conjugates. As shown in FIG. 34(b), thesolid tumor was also discernible from the surrounding tissues with⁶⁴Cu-DOTA-Y-PNA50S-K4. The tumor was clearly imaged with⁶⁴Cu-DOTA-Y-PNA5-K4 at 1 h p.i. (FIG. 34(c)), but not visible at either4 h or 24 h p.i. Contrarily, ⁶⁴Cu-DOTA-Y-PNA7-K4 was able to give afairly good image of tumor at 24 h p.i. (FIG. 34(d)), but not at theearlier time points.

The microPET images were analyzed by the quantification of radiotraceraccumulation in the regions of interest (ROIs). The time-activity curvesof the four PNA conjugates in kidneys (represented by the left kidney),liver, and tumor were shown in FIG. 36. It is apparent that thequantitative data are in agreement with the microPET images (FIGS. 34and 35). Kidneys had the highest concentrations of all four compounds asshown by the standard uptake values (SUVs). The ⁶⁴Cu-labeled conjugatesof PNA50, PNA50S and PNA5 showed slow clearance from kidneys andsignificant accumulation in liver from 1 h to 24 h p.i. While thesethree compounds exhibited similar uptake and retention in kidneys andliver, ⁶⁴Cu-DOTA-Y-PNA7-K4 demonstrated significant different behavior.In kidneys, it showed the lowest uptake and the most efficient clearanceout to 24 h p.i., whereas its liver concentration was the highest, whichwas around five times those of other compounds at 1 h p.i., andmaintained about the same level out to 24 h p.i.Copper-64-DOTA-Y-PNA50-K4 showed the highest SUV (0.076±0.009) in tumor,followed by ⁶⁴Cu-DOTA-Y-PNA5-K4 (SUV: 0.034±0.003) and⁶⁴Cu-DOTA-Y-PNA50S-K4 (SUV: 0.018±0.004) at 1 h p.i. While⁶⁴Cu-DOTA-Y-PNA50-K4 exhibited rapid washout from tumor out to 4 h p.i.,the accumulation level of ⁶⁴Cu-DOTA-Y-PNA50S-K4 remained steady within24 h p.i. Consistent with the microPET images (FIG. 34 d), anappreciable amount of ⁶⁴Cu-DOTA-Y-PNA7-K4 was accumulated in tumor at 24h p.i (SUV: 0.031±0.003).

Biodistribution data of post imaging. The post imaging biodistributionexperiments were carried out right after the microPET imaging at 24 p.i.The concentrations of the four PNA conjugates in blood, liver, kidneys,muscle, and tumor were represented by percent of injected dose per gramof tissue (% ID/g) as shown in FIG. 37. Copper-64-DOTA-Y-PNA50-K4exhibited the highest concentration in kidneys (280±35% ID/g), followedby ⁶⁴Cu-DOTA-Y-PNA5-K4 (251±50% ID/g), ⁶⁴Cu-DOTA-Y-PNA50S-K4 (174±54%ID/g), and ⁶⁴Cu-DOTA-Y-PNA7-K4 (143% ID/g). In liver, while⁶⁴Cu-DOTA-Y-PNA50-K4, ⁶⁴Cu-DOTA-Y-PNA50S-K4 and ⁶⁴Cu-DOTA-Y-PNA5-K4showed similar low concentrations (5.6±0.3% ID/g, 5.2±2.2% ID/g, and7.7±1.1% ID/g, respectively), the accumulation of ⁶⁴Cu-DOTA-Y-PNA7-K4was much higher (28% ID/g). This observation is consistent with thequantification results of the microPET images. Although⁶⁴Cu-DOTA-Y-PNA50-K4 and ⁶⁴Cu-DOTA-Y-PNA5-K4 showed similar tumor uptake(1.50±0.11% ID/g and 1.35±0.11% ID/g, respectively), the tumor/muscle(T/M) ratio of the former (T/M: 6.6±1.1; n=2) is about five times thatof the latter due to the high concentration of ⁶⁴Cu-DOTA-Y-PNA5-K4 inmuscle as shown in FIG. 38. The T/M ratios of ⁶⁴Cu-DOTA-Y-PNA50S-K4(3.0±0.2; n=2) and ⁶⁴Cu-DOTA-Y-PNA7-K4 (2.75; n=1) are in between.Copper-64-DOTA-Y-PNA50-K4 also demonstrated the highest tumor/bloodratio (T/B: 3.4±0.0; n=2) among the ⁶⁴Cu-labeled PNA conjugates.

Discussion

Our approach to the design of cell-specific imaging agents is based on(1) the identification of an abundant unique or uniquely overexpressedmRNA in the target cell (2) the selection of a high affinity antisensePNA for the target mRNA, (3) covalent attachment of a permeation peptidethat allows for reversible cell entry, and (4) covalent or high affinityattachment of positron-emitting radionuclide with high specificactivity. In our approach, the biological function of the mRNA isirrelevant, all that matters is that it is uniquely overexpressed andabundant in the target cell. As such this approach differs from otherantisense approaches that often target oncogenes, which are only presentin low copy number (<100). For this initial study we used SAGE analysisto identify the uniquely overexpressed (approximately 10-fold) andabundant unr mRNA in the MCF-7 cancer cell line (approximately 5000copies/cell), and an RT-ROL assay to identify high affinity antisensebinding sites on the mRNA. PNAs complementary to these sites wereconjoined to the recently described permeation peptide Lys₄ and thehighest affinity PNAs were conjoined to the ⁶⁴Cu binding ligand DOTA tocreate the MCF-7 cell-specific PET imaging agent.

Antisense oligonucleotides have been reported as nuclear imaging probes(PET and SPECT) in forms of unmodified oligomers or viral/non-viralcarrier-oligomer conjugates. While the carrier systems couldsignificantly enhance the tumor accumulation of oligonucleotides due tothe increased payload of the carriers, a major concern is how to addresswhether the biodistribution is determined by the carrier or theoligomer. This method may also encounter the common difficulties of drugdelivery systems, such as sequestration by reticulo-endothelial system(RES) and immunogenicity of the carriers. It is self-evident thatunmodified or minimally modified oligonucleotides should be studiedin-depth before they could be applied to the gene transfection or drugdelivery systems for various proposes.

As a special class of oligonucleotide analogs, the biological behaviorof peptide nucleic acids has been extensively studied in vitro and invivo since they were first proposed. In contrast, the utilization ofPNAs as imaging probes of cancer for PET and SPECT is very limited,probably because the accumulation of the PNA imaging probes in thetumors could barely reach a detectable level. Recently, Sazani et al.(Nat. Biotechnol., 2002, 20, 1228-1233) reported that a PNA with fourlysine at its N terminus exhibited superior in vivo properties andsequence-specific activity compared to its analog with one lysine.Therefore, we introduced the four-lysine tail to our unr mRNA targetingPNAs and further incorporated a DOTA chelating moiety for ⁶⁴Cu microPETimaging and tissue distribution studies.

The labeling of DOTA-Y-PNA-K4 conjugates with ⁶⁴Cu was straightforward.The radiochemical yields were reasonably high, and nearly perfectradiochemical purity was achieved after DTPA challenge ofnon-specifically bound ⁶⁴Cu and centricon separation. The specificactivity of the ⁶⁴Cu-labeled PNA conjugates was ca. 200 mCi/μmol (7,400MBq/μmol) at the injection time, which is the highest specific activityever achieved for radiolabeled oligonucleotides to the best ofknowledge. The radiolabeling conditions were optimized from the trialsvarying reaction temperature, time, pH, and media (data not shown).

Concerned that our PNA conjugates might have rapid in vivo kinetics, wecarried out biodistribution experiments in normal mice via i.v. and i.p.modes. The biodistribution via i.v. was performed within a short timeperiod (4 h) post injection, while the i.p. experiment was conducted at24 h and 48 h p.i. All the four PNA compounds showed high kidney uptakeand long retention throughout time (FIGS. 32 and 33), no matter theinjection mode. This observation is in agreement with McMahon's reporton the in vivo behavior of PNAs (McMahon et al., Antisense Nucleic AcidDrug Dev, 2002, 12, 65-70). While both PNA conjugates with unr mRNAantisense (PNA50) and sense (PNA50S) sequences exhibited a very similarpattern of tissue distribution within 1 h p.i., the conjugate with sensesequence showed a remarkably higher accumulation in kidney than itsantisense counterpart (P<0.005) at 4 h p.i. (FIG. 32) which must berelated to the differences in their base composition or sequence. As aresult of the rapid pharmacokinetics of PNAs, our PNA conjugates showeda very low level of uptake in blood (<4% ID/blood) at the first timepoint of the i.v. biodistribution (20 min p.i.), and efficient clearanceafter then (FIG. 32). The liver uptake was only around 5% ID/organ,which is at the similar level of the reported PNAs. The i.p.biodistribution showed the same profile as the i.v. experiment (FIGS. 32and 33), which indicates that the tissue distribution of the PNAconjugates cannot be significantly altered by the administration modes.Interestingly, the PNA conjugates with sense sequence exhibited asignificantly higher muscle accumulation at 48 h p.i. as compared to itsantisense analog (P<0.002). It is possible that the sense PNA iscomplementary to the same or similar sequence found in mouse muscle, orthat it shows some preferential affinity to some other biomolecule inmuscle.

As shown in the microPET images (FIG. 34), the tumor with the size of100-320 mg in CB-17 SCID mice can be imaged with the four ⁶⁴Cu-labeledPNA conjugates. This is probably because of their high specificactivity, which is about 5 times higher than that of the most recentlyreported ⁶⁸Ga-labeled oligonucleotide (Roivainen et al., J. Nucl. Med.,2004, 45, 347-355). However, the image contrast of tumor at differenttime points varies greatly with different compounds. At 1 h and 4 hp.i., the tumor was not detectable with ⁶⁴Cu-DOTA-Y-PNA7-K4, whereas itgave a good tumor contrast at 24 h p.i. (FIG. 34(d)). Interestingly,⁶⁴Cu-DOTA-Y-PNA5-K4 behaved in the opposite way: it can only image thetumor at 1 h p.i., not at either 4 h or 24 h p.i. (FIG. 33(c)). Both⁶⁴Cu-DOTA-Y-PNA50-K4 and ⁶⁴Cu-DOTA-Y-PNA50S-K4 showed the tumor image atall the three time points, but the former conjugate exhibited a muchhigher tumor to background contrast (FIGS. 34(a) and 34(b)). Thequantitative analysis was performed on the microPET images, and theresults are in good agreement with the post imaging biodistribution data(FIGS. 36 and 37). This further confirmed the quantitation capability ofmicroPET techniques in the field of molecular imaging.Copper-64-DOTA-Y-PNA5-K4 exhibited a similar tumor uptake to⁶⁴Cu-DOTA-Y-PNA50-K4, however, the tumor was not discernible from thesurrounding tissue at 24 h p.i. due to its relatively high accumulationin muscle and blood (FIG. 34). This observation clearly demonstratesthat the rapid and efficient clearance from blood and muscle is one ofthe essential criteria for a good imaging agent besides the high uptakein target organs. To the best of our knowledge, ⁶⁴Cu-DOTA-Y-PNA50-K4 hasshown the highest and reproducible tumor/muscle ratio both at 4 h(7.9±3.3, data not presented) and at 24 h p.i. (6.6±1.1) among thereported non-carrier bound oligonucleotides in the detection of cancerin vivo via nuclear imaging modalities. Further modification of theconjugate of DOTA-PNA50 is under investigation to minimize the kidneyaccumulation while maintaining the tumor-targeting property.

In summary, we have designed and synthesized four PNA with a four-lysinetail at the C terminus, one with a sense sequence and three withantisense sequences for the unr mRNA, which is overexpressed in MCF-7cell line. In order to evaluate their in vivo behavior andtumor-targeting property, we incorporated a DOTA-moiety into the PNAs sothat the conjugates can be radiolabeled with ⁶⁴Cu for biodistributionand microPET imaging studies in normal and MCF-7 tumor bearing mice. Ofthe PNA conjugated studied, ⁶⁴Cu-DOTA-Y—PNA50-K4 showed the best imagecontrast of MCF-7 tumor in CB-17 SCID mice, and its tumor/muscle ratiois reproducible and the highest among the radiolabeled oligonucleotidesthat have been reported for in vivo detection of tumor. Our studiesfurther indicate that antigene and antisense PNAs have great potentialto be developed as oncogene- or mRNA-specific probes for early diagnosisof specific cancers. In addition, such antisense ⁶⁴Cu-PNA constructs mayalso be able to function as patient specific therapeutics because of thedecay characteristics of ⁶⁴Cu when delivered in higher amounts.

EXAMPLE—SET E

Targeting MCF-7 Cells With Antisense PNA'S to Uniquely Overexpressed UNRmRNA

We discovered a uniquely overexpressed and highly abundant mRNA specificto a cell and successfully used that mRNA as an internal receptor for anantisense PNA molecule that showed it was capable of reversibly enteringcells.

We discovered that our method is advantageous for targeting MCF-7 breastcancer cells which uniquely overexpress a very abundant unr mRNA(upstream of N-ras or N-ras related gene). Antisense binding sites onthe unr mRNA were mapped by application of an improved RT-ROL assay, anda newly developed SAABS (serial analysis antisense binding sites) assay.The relative affinity of ODNs complementary to the antisense bindingsites was obtained by a newly developed dot-blot assay. Dissociationconstants for tight binding ODNs identified through the dot blot assaywere obtained by a newly developed Dynabead assay. Hybrid PNAscorresponding to the ODNs with the highest affinities were synthesizedwith an N-terminal CysTyr and C-terminal Lys₄ sequence and their bindingquantified by the Dynabead assay. Hybrid PNAs with K_(d)'s of approx. 10pM for unr mRNA were identified in this manner and shown to bind to unrmRNA extracted from MCF-7 cells by a PCR assay. Two fluorescentlylabeled PNAs with the NLS (nuclear localization sequence) permeationpeptide were shown to selectively bind to MCF-7 cells in vitro by bothfluorescence microscopy and a bulk fluorescence assay, therebyvalidating this antisense approach for cancer cell targeting.

Identification and Selection of a cancer-specific overexpressed MRNA.

To validate the antisense cell targeting approach, we searched the NCBISAGE database (http://www.ncbi.nlm.nih.gov/SAGE/) for abundant mRNAsthat are >10-fold overexpressed in standard cancer cell lines comparedto any normal cell lines. In doing so we discovered that the wellstudied MCF-7 breast cancer cell line contains an almost 10-fold higheramount of the unr mRNA (GI: 20070240), or upstream of N-RAS otherwiseknown as the NRAS-related gene, than normal cell lines, and is presentat a level of about 5,000 tags/million, or roughly 5,000 copies percell. We found a full length cDNA clone from the I.M.A.G.E. consortium(5285557 from the NIH_MGC_(—)96 library, UniGene Libary 6001), andobtained the pBluescriptR vector containing the cDNA through the ATCC(American Tissue Culture Collection, #7020864). We were able tosuccessfully produce the unr mRNA in high yield and homogeneity by invitro transcription with T7 RNA polymerase. The unr mRNA was recoveredfor use as reported herein.

RT-ROL Assay. To identify the sites on the unr mRNA that could bindPNAs, we adopted and improved a mapping method based on using randomoligonucleotides attached to a PCR tag to prime reverse transcriptionfollowed by PCR amplification. In the original method priming end was acompletely random sequence, making it difficult to precisely assign thePCR products to a specific sequence. To get around this problem wesynthesized a set of four separate primers each containing a PCR tag anda random 9-mer terminating in unique nucleotide (FIG. 18) thereby makingit much easier to assign the PCR product bands to specific sequences.The entire unr mRNA was mapped in this way using 7 forward primers(unrX) for each section of the transcript and the PCR tag on the randomoligodeoxynucleotide library (FIG. 19(A)). A number of discrete PCRbands were observed (FIG. 19) which were then analyzed to reveal the ODNbinding sites (FIG. 20). Approximately 50 antisense sites on the unrmRNA were identified by this RT-ROL assay.

Serial Analysis of Antisense Binding Sites—SAABS. A potential problemwith the RT-ROL assay is that that priming of reverse transcriptionmight be sensitive to secondary structure, and as a result, theintensity of the PCR bands produced in the RT-ROL assay might notcorrespond to the actual binding affinity. To eliminate this problem wedeveloped an enzyme independent method for mapping antisense bindingsites that adapts methodology used in SAGE (Serial Analysis of GeneExpression) to determine the sequence and relative frequency of membersof a random library of ODNs that bind to an mRNA molecule.

The overall procedure is shown in FIG. 21, and consists of incubating arandom ODN library linked to two PCR tags with a RNA molecule that isbound to a Dynabead. The PCR tags are prevented from hybridizing to themRNA by binding to the complementary ODNs CS I and CS2. Followingincubation with the mRNA, the Dynabeads are spun down and washed toremove unbound ODNs. The bound ODNs are then PCR amplified withbiotinylated primers Bio-S1 and Bio-S2, restricted to give 12-mers,separated from the cleaved PCR tags by strepavidin, concatenated byligation, cloned into the pZero plasmid, and transfected into E. coli.Plasmids containing inserts are then sequenced, and the sequence of theantisense ODNs are extracted from between the restriction sites andmatched to their complementary site on the RNA sequence by a computeralgorithm. The relative frequency of the antisense sequences found boundto the unr mRNA is plotted against mRNA position in FIG. 22. Some ofthese sites had already been detected by the RT-ROL assay. Fifteen ofthe highest frequency sites were chosen for further analysis.

Dynabead Dot Blot Assay. To determine the relative affinity of ODNs forthe antisense binding sites determined by the RT-ROL and SAABS assays,we developed a sensitive and semi quantitative dot blot assay. In ourfirst attempts we used a standard protocol that involvesphotocrosslinking of antisense ODNs to a nylon membrane and thenquantifying the amount of radiolabeled mRNA that becomes bound.Unfortunately, this technique did not appear to be very reproducible,probably because of the non-uniformity of UV crosslinking of ODNs ofvarying sequence to the nylon membrane.

In developing a better dot blotting method for screening ODN bindingsites, we investigated the use of Dynabeads to anchor the RNA to theblot. We found that the binding capacity of the streptavidin coatedbeads for biotinylated unr mRNA produced by transcription in thepresence of Bio-dUTP, to be about 1 pmol of RNA/20 μL of Dynabeads (FIG.23A) and that the Dynabead bound mRNA could bind about one half anequivalent of a radiolabeled ODN under saturating conditions (FIG.23(B)).

The Dynabeads were then used to bind the mRNA to the nylon membrane inthe dot blot assay (FIG. 23(C) and FIG. 23(D)) to assess the relativebinding affinities of 20-mer ODNs for binding sites identified by theRT-ROL and SAABS methods that we had previously reported. ODNs for thequantitative assays were chosen from both high affinity binding sitesand low affinity binding sites and the sequence optimized by shifting tothe 5′- or 3′-end to minimize self-complementarity and shortened to15-mers to minimize unfavorable electrostatic interactions (indicated byx-2 in the code for the ODN, where x=the parental ODN). On the basis ofthis assay, about 15 ODNs were selected for more quantitative asssays.

Dynabead ODN Binding Assay. Initially we attempted to obtain ODN bindingconstants to the unr mRNA by a Centricon centrifugal filtration assay.In this assay 5′-³²P-radiolabeled ODN incubated with increasingconcentrations of mRNA, and bound ODN is separated from free ODN bycentrifugation through a filter that does not allow the mRNA, and anyODN-bound mRNA to pass through the filter. Unfortunately, this methoddid not work very well due due to problems with non-specific binding thefilter. To get around this problem, we developed a new Dynabead-basedassay method which makes use of a magnetic field to separate moleculesbound to a Dynabead from free molecules in solution without the use of afilter (FIG. 24). The unr mRNA was attached by using streptavidin coatedDynabeads and biotinylated RNA produced by transcription in the presenceof Bio-dUTP. The Dynabead method gave much better results than theCentricon-based method, though it appeared that there was a variableamount of non-specific binding which resulted in measurableradioactivity in the bound fraction at low concentrations of mRNA no ODNshould be bound (FIG. 25). This non-specific binding could be easilysubtracted during the non-linear least squares fitting used to determinethe dissociation constants (see testal section). As can be seen in Table1, 5 ODNs show K_(d)s of 1 nM or less, and 9 have K_(d)s of less than 3nM.

Design and Synthesis of the antisense PNAs. Because ODNs are readilydegraded in vivo and additionally cause cleavage of the mRNA transcriptto which they are bound via RNase H enzyme activity, they are notsuitable for in vivo targeting. Peptide nucleic acid (PNA) is ideallysuited for this purpose because it is a nucleic acid analog with apeptide backbone in place of a sugar phosphate backbone that is highlyresistant to degradation, does not activate RNase H activity and alsohas high affinity for complementary mRNA. We therefore designed hybridPNAs corresponding to the 4 tightest binding ODNs, and ODN 5-2 which isless tightly bound. A sense sequence corresponding to PNA50-2S was alsomade as a control. The PNAs were synthesized with 4 lysines at thecarboxy terminus (FIG. 10), as a permeation peptide for in vitro and invivo studes in mice, and to aid in water solubilization and in mRNAbinding. A cysteine—tyrosine sequence was added to the amino terminalend to enable attachment of reporter groups to the cysteine, andradioiodination of the tyrosine. The PNAs were synthesized by standardsolid phase Fmoc chemistry on an ABI Expedite 8909 automatedsynthesizer, purified by reverse phase HPLC, and characterized bymolecular weight determination by MALD-TOF (Table 2).

PNA binding affinity. The binding affinity of the PNAs for the unr mRNAwere determined by monitoring bound and free ¹³¹I-labeled PNA as afunction of mRNA concentration using the Dynabead method that wedeveloped for the ODNs (FIG. 24). We chose ¹³¹I to label the PNAsbecause of the high specific activity in which it can be obtained andits short half-life of 8 days. The PNAs were radiolabeled by a publishedprocedure previously used to label PNAs with ¹²⁴I utilizing chloramine-Tas an oxidant (FIG. 26). We also checked the integrity of the PNAproduced under theses conditions by repeating the labeling test with ashort PNA test sequence, DOTA-Tyr-ATGC-Lys with non-radioactive iodineby the chloramine-T method and also by IODO-beads and analyzed theproducts by MALDI. We found that at 5 min reaction time, both methodslead primarily to the mono-iodinated compound and some of thediiodinated compound (tyrosine can react twice), but that at longertimes, other products are produced (FIG. 27). Thus the 5 min time periodappears to be optimal for radiolabeling by I-131.

In the first set of PNA binding tests we discovered that we were notrecovering the entire radioactivity. We were able to trace this tonon-specific binding of the PNA to the standard polyethylene microfugetubes. After ordering and testing a number of tubes and microtiterplates advertised as minimizing binding of peptides and nucleic acids,we found that the Corning NBS microtiter plates had the lowest bindingaffinity. Individual tubes were obtained by sawing the plates intopieces. Triplicate sets of data were obtained and a plot of % bound as afunction of RNA concentration was fit to a simple two state bindingequilibrium as we had done for the ODNs (FIG. 28). The K_(d)s for thePNAs and the corresponding ODNs are tabulated in Table 3. As expected,the PNAs show very high binding affinity (low K_(d)s) for the testedsites on the unr mRNA with K_(d)s that range from 7 to 50 pM at 0.1 Msalt.

The binding affinities of the PNAs for the sites are much greater (lowerK_(d)s) than that of the corresponding ODNs. The difference is bindingaffinity under physiological conditions is likely to be much greaterthan it would appear from the tabulated data since the PNA dissociationconstants were obtained at the physiological concentration of 0.1 MNaCl, whereas the ODN dissociation constants were acquired at 1 M salt.At 0.1 M salt the K_(d)s for the ODNs are expected to be greater due toelectrostatic repulsions, whereas the PNAs are expected to have lowerK_(d)s due to favorable electrostatic attraction with the Lys₄ tail. Thecontrol PNA, PNA 50-2S which is identical in sequence to the mRNA targetshowed no significant binding in the range of RNA concentrations thatbound tightly to the antisense sequences.

Binding of PNA50-2 to unr mRNA isolated from MCF-7 cells.

To determine whether or not the 50-2 antisense binding site identifiedby the RT-ROL and SAABS assays on unr mRNA produced by T7 RNA polymerasein vitro exists on unr mRNA produced by MCF-7 cells in vivo we devised asimple RT-PCR assay. For this assay, total RNA from MCF-7 is subjectedto RT-PCR with various combinations of primers as illustrated in FIG.29. If reverse transcription takes place from either the 50-2 site withODN50-2 or the polyA tail with a dT₁₈ primer, and extends to the end ofthe mRNA, one should get a 439-mer PCR product when one uses the two PCRprimers UNR12-1 and UNR12-2. Indeed, if dT₁₈ is used as a primer, (LaneA of FIG. 29) a PCR product is indeed observed, as it is when ODN50-2alone is used as a primer (lane B), demonstrating that this site isaccessible in unr mRNA produced by MCF-7 cells. When PNA50-2 alone isused no product is visible as PNAs cannot serve as primers for reversetranscriptase (Lane C). When ODN50 is used in the presence of PNA50, noPCR product is again observed, which is consistent with the much tighterbinding affinity of PNA than DNA for the same site on the mRNA (Lane D).When PNA50 is present during extension by dT₁₈ no PCR product is seensuggesting that PNA50 binds so tightly that it can block reversetranscription past this site (Lane E). When the sense PNA, PNA50S isincubated with ODN50 no PCR product is observed which is consistent withduplex formation between PNA50S and ODN50 (Lane F). When the samereaction is carried out in the presence of dT₁₈, a PCR product isobserved (Lane G) as expected since there is nothing to block reversetranscriptase from elongating dT₁₈. Together, the results of these testsconfirm that both ODN50 and PNA50 bind to the unr mRNA produced by MCF-7cells, and furthermore that PNA50 binds much more tightly than ODN50, asbourne out by results of the binding assays.

Binding of Fluorescently labeled PNAs to MCF-7 cells. Initially weexpected that Cys-Tyr-PNA-Lys₄ hybrid PNAs would be able to enter cellsin vitro because of the prescence of the Lys₄ permeation peptide andthat we could study this process by labeling the cysteine with afluorescent reporter group. Unfortunately, we found that the fluoresceinlabeled PNAs formed aggregates and did not enter the cells, whereas afluorescein labeled CysArg₉ peptide did. To see if changing thepermeation peptide would help with the cell culture targeting tests wesynthesized a series of fluorescently PNAs with the nuclear localizationsequence (NLS), KPKKKRKV (Table 2) following, which contains anadditional lysine and arginine, along with a proline and valine. WhenMCF-7 cells were incubated with 1.0 μM Flu-PNA-NLS for 4 h, 24 h and 48h, and then washed 5 times with PBS prior to fixation withparaformaldehyde, PNA50-2 and PNA7 showed that highest fluorescencestaining of the cells, whereas PNA5-2 and PNA50-2S showed comparitivelylittle (FIG. 30). To get a more quantitative assessment of the targetingcapabilities of the PNAs, the MCF-7 cells were treated as above with thefluorescein labeled PNAs for 24 h. Following this the cells were lysedand the fluorescence of the mixture was measured at 520 nm withexcitation at 488 nm on a SPEX fluorimeter. In this assay, PNA7 showedthe highest binding with about 12-fold higher fluorescence that thecontrols (FIG. 31). PNA50-2 was next best with 4-fold higherfluorescence. PNA5-2 did not show any significant binding, despitehaving show a high affinity for unr mRNA in vitro.

Overall

We have developed and successfully used a modified RT-ROL and a newSAABS methodology for identifying antisense binding sites on mRNAproduced in vitro, and Dynabead-based assays for determining the bindingaffinity of antisense ODNs and PNAs for these sites. Furthermore, weshowed that antisense PNAs against the unr mRNA which is abundantlyoverexpressed in the MCF-7 breast cancer cell line concentrates withinthese cells when attached to the NLS permeation peptide, whereas anon-complementary sequence did not.

Materials and Methods

Image clone 5285557 containing the unr (upstream of N-ras) mRNA sequence(D1S155E, NM_(—)007158.2, GI: 20070240, ATTC clone #7020864). pZeroplasmid for SAGE analysis is from Invitrogen. The MCF-7 cell line wasobtained the Washington University Medical School in St. Louis.5(6)-carboxyfluorescein succinimidyl ester (5(6)-FAM SE) was purchasedfrom Molecular Probes, diisopropylethylamine (DEA), trifluoroacetic acid(TFA), m-cresol and diethyl ether (anhydride) were purchased fromAldrich. α-N-9-Fluorenylmethoxycarbonyl (Fmoc) protected amino acids(Val-OH, Pro-OH, Lys(Boc)-OH and Arg(Pbf)-OH) were purchased fromNovaBiochem.O-(7-Azabenzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU), Fmoc-XAL PEG-PS resin, PNA building blocks(Fmoc-A-(Bhoc)-OH, Fmoc-C-(Bhoc)-OH, Fmoc-G-(Bhoc)-OH and Fmoc-T-OH) andother reagents and solvents for PNA and peptide synthesis were purchasedfrom PerSeptive Biosystems. Fluorescence spectra were recorded on SPEXFluoromax instrument. UV spectral data were acquired on a Bausch andLomb Spectronic 1001 spectrophotometer or Varian Cary 100 Bio UV-VisibleSpectrophotometer. Matrix-assisted laser desorption ionization (MALDI)mass spectra of PNA-peptide conjugates were measured on PerSeptiveVoyager RP MALDI-time of flight (TOF) mass spectrometer using sinapinicacid as a matrix and calibrated versus insulin (average [M+H⁺]=5734.5)that was present as an internal standard. High-pressure liquidchromatography was carried out on Beckman Coulter System Gold 126 withMicrosorb RP C18 column (300 Å pore, 5 μm particle size, 4.6×250 mm)using 1 mL/min linear gradients of solvent B (0.1% TFA in acetonitrile)in A (0.1% TFA in water). Peptide-PNA conjugates were quantified byspectrophotometric A260 values.

Successful production of unr RNA. Transcription reactions were carriedout using the Promega RiboMAX Large Scale RNA production systemutilizing the T7 promoter at the 5′-end of the unr gene in the IMAGEclone and cleaving the clone at the unique NdeI site at the 3′-end ofthe gene prior to transcription. Transcription was allowed to proceedwith T7 RNA polymerase and NTPs for 3 h at 37° C., and was followed byDNase treatment, LiCl precipitation, phenol extraction, and ethanolprecipitation. NTPs were then removed with a NucAway spin column.Radiolabeled RNA was produced with [α-³²P]-UTP. The final purified unrRNA is quite homogeneous and corresponds in size to the expected 3.4 Kbtranscript. For the Dynabead assay, the unr mRNA was transcribed in thepresence of Bio-dUTP.

RT-ROL mapping of antisense sites on unr RNA. The ODNs for mapping theantisense binding sites on the unr RNA by the modified RT-ROL assay areshown in FIG. 35. The mapping was carried out by mixing the RNA andrandom oligo library at 65° C. for 5 min with dNTPs followed by aquickly chilling in ice and then adding ribonuclease inhibitor, heatingto 24° C. for 10 min, then 42° C. for 2 min and then adding SuperScript,and then incubating at 42° C. for 50 min, then inactivating by heatingat 70° C. for 15 min. Then 5′-endlabeled PCR primer, dNTPs, and Taqpolymerase was added and subjected to 30 cycles of PCR (94° C.-1 min,55° C. 1 min, 72° C. 1 min, 72° C. 10 min, 4° C.). The products werethen run on 6% denaturing polyacrylamide gels.

SAABS Method for Mapping Antisense Sites on unr mRNA. Dynabeads(Dynabead®M-280streptavidin, DynalBiotech) were first washed withsolution A (DEPC-treated 0.1 M NaOH, DEPC-treated 0.05 M NaCl), followedby three times with solution B (DEPC-treated 0.1 M NaCl), and thenwashed two times with 100 μL of hybridization solution (1M NaCl,Tris-HCl pH 7.4). The beads were then resuspended in 50 μl ofhybridization solution. The RNA (3 μL of 3 μg/μl) was incubated with 2μl of CRNA-Bio (4.7 μg/μl) and of 45 μl of hybridization buffer at 65°C. for 5 min, and then cooled at room temperature for 10 min. The RNAsolution was then added to a suspension of the beads at 25° C. for 20min. The beads were then washed three times using hybridization buffer,and then resuspended in 50 μl of hybridization solution. The ROL ODN (5μl) were then annealed with the complementary ODNs CS1 (12 μl), and CS2(8.6 μl) in hybridization buffer (23 μl) by heating at 65° C. for 5 min,and then cooled to room temperature for 10 min. The RNA solution wasthen mixed with S1-ROL-S2 together with 2 μl RNasein inhibitor, andincubated at 27° C. for 1 h. During the incubation, the beads wereresuspended every 15 min by pipetting. Following the incubation, thebeads were washed 6 times with the hybridization solution and thenresuspended in 100 μl of H₂O. Thirty cycles of PCR were carried out withTaq DNA polymerase, and 5′-biotinylated S1 and CS2 primers. Afteramplification, the reaction mix was directly loaded onto a 12%denaturing PAGE gel and electrophoresis was carried out at 10 V/cm for 4hours. The 50 bp band was excised and purified by use of Strepavidinbeads. The 50 bp PCR product was directly digested with Nla III at 37°C. overnight to release the tags. The digested product was loaded onto4% agarose gel and run at 10 V/cm. After staining the gel with 0.25μg/ml ethidium bromide, the 12 bp band was excised. Ligation of the12-mers was carried out at 16° C. for overnight. Concatemers wereisolated using the Qiaquick Gel Extraction Kit (Qiagen), following themanufacturer's manual. The pellet was dissolved in ligation buffer andligation was carried out with T4 DNA Ligase and ATP at 16° C. for 2 h.The mixture was then electrophoresed on a 1% agarose gel (TAE) andfractions with 300-500 bp length were excised. Concatemers were ligatedinto the SphI site of pZero-1 (Invitrogen) with T4 DNA ligase and ATPfor 4 h at 16° C. and then transfected into E. coli, following themanufacturer's manual (Oneshot®Top10, invitrogen). The transfectantswere plated on low salt LB plates containing 50 μg/ml Zeocin™ andincubate for about 18 h at 37° C. Zeocin™ resistant transformants werepicked using pipette tips, incubated in 5 ml SOB containing 50 μl/mlZeocin™ and grown overnight at 37° C. Plasmid DNA was prepared by theRapid method (Molecular Clone) and Plasmid min prep kit (Qiageon).Digest DNA using XbaI and HindIII, analyze digestion in 1% agarose gel.Plasmids containing sizable inserts were then forwarded for sequenceanalysis using the Sp6 primer.

ODN Dynabead Binding Assay. The radiolabeled ODN (100 pM) was incubatedwith the 0.003 nM-10 nM of biotinylated mRNA and 1 μL of Rnase inhibitorfor 4 h at 37° C. in a total volume of 100 μL. Then Dynabeads M-280Steptaviden (Dynal Biotech) were added, 25 μL for 10 nM RNA, 12.5 μL for3 nM RNA and 5 μL for all other concentrations of RNA, and mixed for 30min. The beads were separated by a magnet and the solution removed. Thebeads were resuspended in 100 μL of water and both solutions counted byliquid scintillation to give free and bound fractions directly.

ODN Binding Data Analysis. A plot of fraction bound vs. mRNAconcentration was fitted by non-linear least squares fitting with theKalaidagraph program to the following analytical curve:$F_{B} = {C + {\frac{\begin{matrix}{\left( {1 - C} \right)*\left( {\left( {\lbrack{ODN}\rbrack + K_{d} + \lbrack{RNA}\rbrack} \right) -} \right.} \\\sqrt{\left( {\lbrack{ODN}\rbrack + K_{d} + \lbrack{RNA}\rbrack} \right)^{2} - {4 \cdot \lbrack{ODN}\rbrack \cdot \lbrack{RNA}\rbrack}}\end{matrix}}{2 \cdot \lbrack{ODN}\rbrack}w}}$here:

C=fraction of ODN non-specifically bound

[ODN]=total ODN concentration

[RNA]=total RNA concentration

K_(d)=dissociation constant

PNA-Lys₄ peptide synthesis and purification. The hybrid PNA-peptideswere synthesized in 2 μmol scale on an ABI 8909 automated DNA/PNAsynthesizer using Fmoc chemistry and the manufacturer's protocols,reagents and PNA monomers. After completion of automated synthesis, PNAswere cleaved from the solid support and the bases were deprotected usingtrifluoroacetic acid:m-cresol (4:1) for 2 h and then precipitated withdiethyl ether. The hybrid PNA-peptides were purified by reverse phasechromatography on a Microsorb-MV 300-5 column, (250×4.6 mm column, 300 Apore sizes, Varian Inc.) C-18 column with a 0-5% B/5 min, 5-60% B/30min, 60-95% B/5 min, 95% B for 5 min, 95%-0%/10 min where A=0.1% TFA inwater, B=0.08% TFA in CH₃CN. The fractions containing the products wereevaporated to dryness in a Savant Speedvac, and redissolved in purewater.

Radioiodination of the PNA-peptides with ¹³¹I. The PNA-peptides wereradioiodinated by a procedure described for iodination of PNAs with¹²⁵I. Thus Na¹³¹I (12 μL, 5 mCi/120 μL solution from Amersham) was addedto 2 μL of water and 2 μL of 1 M sodium phosphate, pH 7, then 2 μL of100 μM NaI was added, followed by 4 μL of 1 mM chloramine-T and 4 μL of100 μM PNA. The reaction was mixed by micropipetting, and allowed tostand for 5 min at room temperature and quenched with 4 μL of 10 mMsodium metabisulfite. The reaction mixture was then diluted with 80 μLof water and placed onto a Waters Sep-Pak cartridge (Vac C18, 6 cc, Part# WAT036905) that had been prewashed with 5 mL of 0.1% TFA in water. Theunreacted Na¹³¹I (average of 27% of the total radioactivity) was washedout with 10 mL of 0.1% TFA in water. This was followed by 5-10 mL of 5%acetonitrile in 0.1% TFA/water, which contained little radioactivity.The radiolabeled PNA-peptide (average of 42% of the radioactivity) waseluted with 5 mL of 40% CH₃CN in 0.1% TFA/water, leaving an average of31% of the total radioactivity on the column. The average recovery ofthe labeled PNA-peptide was 57%. An aliquot of the fraction containingthe labeled PNA-peptide was then diluted down to make a 100 μM stocksolution. The actual concentration was then determined base on theactivity of the solution in comparison to the 5 mL stock solution.

PNA Dynabead Binding Assay. The Dynabead assay was carried out asdescribed above for the ODNs except that Corning non-binding assay tubeswere used that were cut out of Corning® 96 Well White Flat BottomPolystyrene NBS™ Microplates (Corning #3600) which have a polyethyleneoxide-like surface. The binding tests were carried out in triplicate.Specifically, the radiolabeled PNA (13-26 pM) was incubated with the0.1-1000 pM of biotinylated mRNA and 1 μL of RNAse inhibitor for 4 h at37° C. in a total volume of 100 μL of 0.1 M NaCl, 50 mM EDTA, 2 mMcacodylic acid. Then 5 μL Dynabeads M-280 Steptaviden (Dynal Biotech)were added and mixed for 2 h. The beads were separated by a magnet andthe supernatant transferred to a liquid scintillation vial. The beadswere washed with 100 μL buffer three times and the washes transferred tothe first scintillation vial. The beads were then transferred to aliquid scintillation vial by suspending in 100 μL of buffer three times.Liquid scintillation fluid (5 mL of CytoScint plus) was then added andboth scintillation vials counted by liquid scintillation to give freeand bound fractions directly.

PNA Binding Data Analysis. The binding data for three tests was averagedand analyzed by fitting the % bound (% B) vs RNA concentration data tothe analytical expression shown below for a two state bindingequilibrium using a non-linear least squares fitting algorithm inKalaidagraph in which the data was weighted according to their standarddeviations. NSB is the % non-specifically bound (to be fit), SB is the %specifically bound (to be fit), [PNA] is the concentration of PNA used(a constant), [RNA] equals the concentration of RNA, a variable, andK_(d) is the dissociation constant for the PNA (to be fit).$\%_{B} = {{NSB} + \frac{\begin{matrix}{({SB})*\left( {\left( {\lbrack{PNA}\rbrack + K_{d} + \lbrack{RNA}\rbrack} \right) -} \right.} \\\sqrt{\left( {\lbrack{PNA}\rbrack + K_{d} + \lbrack{RNA}\rbrack} \right)^{2} - {4 \cdot \lbrack{PNA}\rbrack \cdot \lbrack{RNA}\rbrack}}\end{matrix}}{2 \cdot \lbrack{PNA}\rbrack}}$

Synthesis of the Flu-PNAs-NLS conjugates. A solid supported NLS peptide(NH₂-KPKKKRKV-) was synthesized on a 2 μmol scale by manual benchtopFmoc off synthesis on the universal support XAL-PEG-PS resin.Deprotection was carried out with 20% piperidine in DMF (v/v) and thecoupling step was conducted in presence of N-Fmoc amino acid, HATU (4.0eq) and DIEA (8 eq) in DMF. Capping was conducted with 5% aceticanhydride and 6% lutidine in DMF, alternate washing was applied betweeneach procedure with methanol and DMF, after sequential amino acidcoupling cycles finished, the resulted peptide-resin was washed with DMFand DCM and loaded in Expedite 8909 Synthesizer (Applied Biosystems)without deprotection. The PNAs-peptide conjugates were synthesizedcontinuously by coupling PNA building blocks on N-terminus of NLSpeptide which is attached on resin under standard automated PNAsynthesis protocol. The cartridge containing NH₂-PNA-peptide-resin wastaken out of the synthesizer after PNA synthesis was complete.5(6)-Carboxyfluorescein succinimidyl ester (10.7 mg, 20 μmol) wasdissolved in 300 μl DMF, after DIEA (11 ul, 60 μmol) was added, themixture was introduced into the cartridge with syringe and push thesolution back and forth to agitate the resin suspension once 10 minduring 1 hour. Then the resin was washed with DMF and DCM and dried bypassing nitrogen. TFA/m-cresol (4:1) was used to cleave the conjugatesand remove the side chain protective groups by treatingFlu-PNA-peptide-resin for 2 h at RT 8-10 fold of ethyl ether was addedinto isolated TFA mixture to precipitate the expected product as yellowsolid. The resulted crude products were purified by reversed phase HPLCon a Microsorb C18 column (300 Å pore, 5 μm particle size, 4.6×250 mm)using 5% to 70% linear gradient of solvent B (0.1% TFA in acetonitrile)in A (0.1% TFA in water) over 65 min at the flow rate of 1 ml/min. Theeffluent was monitored by absorbance at both 260 nm and 440 nm and themajor peaks were collected, concentrated to dryness in vacuo, andcharacterized by MALDI-TOF mass spectrometry.

Assessment of MCF-7 targeting ability of Flu-PNA-NLS conjugates byfluorescent microscopy and a bulk fluorescent assay. The MCF-7 cell linewas obtained from American Type Culture Collection (ATCC), and was grownin Eagle's Minimal Essential medium with Earle's balanced salt solutionand 2 mM L-glutamine (EMEM) (ATCC), supplemented with 0.01 mg/ml bovineinsulin, 10% fetal bovine serum (Gibco), penicillin (20 units/ml) andstreptomycin (20 μg/ml) (Gibco), at 37° C. in a humid atmospherecontaining 5% CO₂. MCF-7 were seeded onto eight-well glass chamberslides (Nunc; Naperville, Ill.), and grown to ˜60% confluence. Theculture medium removed and the cells were washed with PBS and 300 μl offresh culture medium with 1.0 μM Flu-PNAs was added. The cells wereincubated at 37° C. in a humid atmosphere containing 5% CO₂ for 4 h, 24h and 48 h, and then washed five times with PBS. The cells were fixed atroom temperature by addition of 4% (v/v) paraformaldehyde in PBS for 20min, followed by three rinses with PBS. The cells were mounted withfluorescence antifading mounting medium following the recommendedprocedures of the manufacturer (Vector Laboratories, Burlingame,Calif.). Preparations were analyzed by fluorescence microscopy. TheMCF-7 cells were treated as above with fluorescein labeled PNA NLSconjugates for 24 h. Following this the cells were lysed and thefluorescence of the mixture was measured at 520 nm with excitation at488 nm on a SPEX fluorimeter.

Table 1. Binding affinity of the antisense ODNs by the Dynabead assay.The site number “x-y” is given as the nucleotide “x” in the mRNA (startcodon at 448) followed by the length, “y”. The code number “x-y” refersto sequence “x” used for the dot blot tests, and if followed by a 2refers to a modified second generation sequence that overlaps theoriginal sequence). K_(d) Site ODN sequence Code (nM) SD 2851-15TGGTGTGCTTTGTGG 50-2 0.43 0.08  676-15 TTTCCCAGTCCGTCG  7-2 0.6 0.12 901-15 ATCTCCAGTTTCCAG S3-2 0.8 0.1 1414-15 TTTGTCACGTCGGTC S5-2 0.90.2 1145-15 CATTTCTGTCCTTGA 16-2 1.1 0.2 1802-15 CATCCTCAGCCTCCT 26-21.2 0.2  839-15 CACTTCCCCATTACG 13-2 1.9 0.4  727-20ATTCGTTCTTCAGGGAGGAT  9 2.6 0.7  476-15 TATGTCCATTGTTGT  5-2 2.8 0.62020-20 CCAAAATTATCCTTCAGAGT 32 21 5 1396-20 TCTGTTGAAATATTAAACCT 21 261.5 1927-15 CCTCTGTTTGTCACT 29-2 33 8 2114-15 TGTCCCCCAGTTCCA 35-2 40 141389-20 AATATTAAACCTAACATGGT 20 73 9.4 2115-15 ATGTCCCCCAGTTCC S7 147120 na CGATTGGAGCGC  V-12 344 148 na AGATCGCAACTCATA  L-15 588 406

TABLE 2 Structure and characterization of PNA conjugates. MW fromMALDI-TOF PNA Conjugate Calc'd Found  5-2Cys-Tyr-CATTATGTCCATTGTTGT-Lys₄ 5641 5648, 5643.4  7-2Cys-Tyr-TTTCCCAGTCCGTCGGTC-Lys₄ 5588 5595.4, 5592.4 50-2Cys-Tyr-TGGTGTGCTTTGTGGATG-Lys₄ 5778.5 5786, 5785, 5781.9 S5-2Cys-Tyr-TAATTTGTCACGTCGGTC-Lys₄ 5651 5657.2, 5650.8, 5667, 5661 S3-2Cys-Tyr-TATCTCCAGTTTCCAGCT-Lys₄ 5571 5580.4, 5571.8 50-2SCys-Tyr-CATCCACAAAGCACACCA-Lys₄ 5561.5 5569.4, 5566.8, 5569.1  5-2Flu-CATTATGTCCATTGTTGT-KPKKKRKV 6353.58 6361.14  7-2Flu-TTTCCCAGTCCGTGGGTC-KPKKKRKV 6136.62 6144.22 50-2Flu-TGGTGTGCTTTGTGGATG-KPKKKRKV 6216.57 6222.19 50-2SFlu-CATCCACAAAGCACACCA-KPKKKRKV 6163.55 6168.93

TABLE 3 Binding affinity of Cys-Tyr-PNA-Lys4 by the Dynabead bindingassay. K _(d) Site PNA Sequence Code (pM) SD 2828-18 TGGTGTGCTTTGTGGATG50-2 21 5  653-18 TTTCCCAGTCCGTCGGTC  7-2 15 4  879-18TATCTCCAGTTTCCAGCT S3-2 7 3 1394-18 TAATTTGTCACGTCGGTC S5-2 50 6  456-18CATTATGTCCATTGTTGT  5-2 22 6 na CATCCACAAAGCACACCA 50-2S >10,000 ndna - not applicable, nd - not determined

EXAMPLE—SET F

Micropet Imaging of MCF-7 Tumors in Mice Via unr mRNA-Targeted PNAsConjugated to Shell Cross-Linked Nanoparticles (SCKs)

In this example the inventors synthesized one targeting and one controlnanoparticle bearing the antisense PNA50 and the sense PNAS sequences,respectively, to image the unr mRNA that is highly and abundantlyoverexpressed in a breast cancer cell line (MCF-7). A DOTA(1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) chelatingmoiety was conjugated to the surface of the nanoparticles so that theycould be radiolabeled with ⁶⁴Cu for biodistribution and microPET imagingstudies in MCF-7 tumor bearing mice. Furthermore, the proteintransduction domain (PTD) of the HIV-1 TAT protein (see SEQ ID 1) wasconjugated for cell membrane crossing. The two nanoparticle weresuccessfully labeled with ⁶⁴Cu under mild conditions and were injectedin CB-17 severe combined immunodeficiency (SCID) mice bearing MCF-7xenografts (ca. 100 mg). MicroPET imaging revealed uptake of bothtargeting and control PNA-conjugated nanoparticles in MCF-7 tumors.However, the uptake of the SCK-PNA50 conjugate was higher compared tothe SCK-PNA50S. Post-imaging biodistribution data comparison (24 h postinjection) revealed a similar tumor specificity for SCK-conjugated andfree PNA50 over PNA50S, suggesting the potential of PNA-conjugated SCKsas tumor specific molecular probes for early detection of cancer andultimately for patient specific radiotherapy.

Test Procedures

SCKs and PNAs were synthisized and purified as reported in Examples setA and E, respectively. PNA50, PNA50S, PTD and DOTA were conjugated onthe surface of the SCKs as reported in Example set B. Radiolabeling with⁶⁴Cu and purification of DOTA-SCK(PTD)-PNA50 and DOTA-SCK(PTD)-PNA50Swere carried out as reported in Example set D. MCF-7 xenografted CB-17SCID mice were obtained as described in Example set D.

MicroPET imaging studies. The microPET imaging studies were carried outusing the microPET® R4 (rodent) scanner (Concorde Microsystems Inc.,Knoxville, Tenn.). MCF-7 tumor-bearing CB-17 SCID mice were anesthetizedwith 1-2% vaporized isoflurane and injected with ca. 100-150 μCi ofactivity in 100 μL saline via the tail vein. At specific time points (1h, 4 h, and 24 h) post injection, the mice were re-anesthetized and thenimmobilized in a supine position on custom-built support beds withattached anesthetic gas nose cones for data collection. After themicroPET imaging at 24 h p.i., the animals were sacrificed andbiodistribution studies were performed. The ratios of tumor to blood(T/B) and tumor to muscle (T/M) were calculated.

Results

The sequences of the antisense and sense PNAs were selected by aprocedure that was described in Example set E. PNA50 with a K_(d) of 21pM for the 2828-18 binding site of the unr mRNA and PNA50S with a K_(d)of >10 nM were selected to be conjugated on the SCK surface to obtain atargeting construct and a control one, respectively. Together with thePNAs, the nanoparticles were conjugated to PTD for cell membranecressing and to DOTA for ⁶⁴Cu-labeling. The two nanoconjugates weresuccessfully radiolabeled with ⁶⁴Cu in 0.1 M ammonium citrate buffer (pH6.5) under mild conditions (3 h at 60° C.).

After DTPA challenge of non-specifically bound ⁶⁴Cu-activity andCentricon-YM100 (MWCO: 100,000 Da) separation, the radiochemical purityof the ⁶⁴Cu-labeled PNA conjugates was nearly 100% as determined byradio-FPLC.

CB-17 SCID mice bearing MCF-7 xenografts were administered of⁶⁴Cu-DOTA-SCK(PTD)-PNA50 and ⁶⁴Cu-DOTA-SCK(PTD)-PNAS via tail veininjection. The mice were imaged at 1 h, 4 h and 24 h post injection,then they were euthanized, the main organs were explanted, weighed andcounted in a gamma counter together with standards to obtain the percentinjected dose per gram tissue (% ID/g) and the percent injected dose perorgan (% ID/organ).

The microPET images in FIG. 39 show the two mice administered with⁶⁴Cu-DOTA-SCK(PTD)-PNA50 (left) and ⁶⁴Cu-DOTA-SCK(PTD)-PNA50S (right)side by side 1 h post injection (Panel A: coronal slice; Panel B:transaxial slice; tumors are indicated by a white solid arrow).Visually, ⁶⁴Cu-DOTA-SCK(PTD)-PNA50 exhibits the highest image contrastof tumor, which is implanted in the nape of the neck.

The post-imaging biodistribution results are consistent with thisfinding and the tumor/muscle and tumor/blood ratios (FIG. 40) confirmthat the antisense PNA (PNA50) maintains a higher target specificitycompared to the control (PNA50S) upon conjugation to the surface of thenano-scale polymeric scaffold (SCK). TABLE 1 Post imagingbiodistribution data in MCF-7 xenograft bearing SCID mice administeredwith ⁶⁴Cu-DOTA-SCK(PTD)-PNA50 and ⁶⁴Cu-DOTA-SCK(PTD)-PNAS (ca. 150μCi/100 μl). The data are presented as percent injected dose per gramtissue (% ID/g) and percent injected dose per organ (% ID/organ) %ID/gram % ID/organ ⁶⁴Cu-DOTA- ⁶⁴Cu-DOTA- ⁶⁴Cu-DOTA- ⁶⁴Cu-DOTA- SCK(PTD)-SCK(PTD)- SCK(PTD)- SCK(PTD)-PNA50 PNA50S PNA50 PNA50S blood 0.46310.4969 0.5382 0.6226 lung 4.2941 2.1310 0.4792 0.2272 liver 4.98793.5876 3.7090 2.9515 spleen 1.6291 1.9820 0.0738 0.0920 kidney 3.26092.3850 0.3858 0.2726 muscle 0.2502 0.2754 1.7028 2.0214 fat 0.48210.2491 1.1044 0.6154 heart 0.8383 0.9867 0.0573 0.0759 brain 0.13610.1291 0.0496 0.0455 bone 1.4135 0.1372 2.5576 0.2677 tumor 1.29790.7534 0.1887 0.1002

Compared to the native SCK, the PNA- and PTD-conjugated nanoparticleshave a lower accumulation in blood (⁶⁴Cu-TETA-SCK: 1.05±0.33% ID/g;⁶⁴Cu-DOTA-SCK(PTD)-PNA50: 0.4631% ID/g; ⁶⁴Cu-DOTA-SCK(PTD)-PNA50S:0.4969% ID/g) and liver (⁶⁴Cu-TETA-SCK: 23.34±3.76% ID/organ;⁶⁴Cu-DOTA-SCK(PTD)-PNA50: 3.7090% ID/organ; ⁶⁴CU-DOTA-SCK(PTD)-PNA50S:2.9515% ID/organ). At the same time, the derivatized SCKs have a loweruptake in kidney at 24 h post injection, as compared to thenon-conjugated PNAs (⁶⁴Cu-DOTA-SCK(PTD)-PNA50: 0.3858% ID/organ;⁶⁴Cu-DOTA-SCK(PTD)-PNA50S: 0.2726% ID/organ; ⁶⁴Cu-DOTA-PNA50-K4:29.95±3.28% ID/organ; ⁶⁴Cu-DOTA-PNA50S-K4: 22.72±8.45% ID/organ). Thecomparison of the biodistribution data in blood, kidney and liver isdepicted in FIG. 41.

In summary, we have designed and synthesized two shell cross-linkednanoparticle bearing PNAs with different affinity for a mRNAoverexpressed in MCF-7 tumor cells (ca. 5000 copies per cell): atargeting SCK conjugated with the antisense sequence PNA50 and a controlSCK conjugated with the sense sequence PNA50s. Upon conjugation to thenanoparticle, the antisense sequence maintained a highest affinity forthe target mRNA compared to the sense sequence, as confirmed by bothmicroPET imaging (FIG. 39) and post-imaging biodistribution (FIG. 40).The MCF-7 tumors accumulated the radiotracers and were clearly visibledespite the decreased bioavailability of the PNA-conjugatednanoparticle, as confirmed by the low blood uptake of⁶⁴Cu-DOTA-SCK(PTD)-PNA50 as compared to the free ⁶⁴Cu-DOTA-PNA50-K4(FIG. 41). At the same time, the PNA-SCK construct exhibited a morefavorable clearance profile from the main excretory organs compared toboth the native nanoparticles and oligonucleotide analogs, as confirmedby the uptake values in kidney and liver (FIG. 41). These findingssuggest PNA-conjugates SCKs are promising tools for imaging of mRNAoverexpressing or uniquely expressing tumors in vivo and ultimately mayallow for the development of effective agents for patient specificradiotherapy.

1. A diagnostic target-specific imaging probe comprising anintracellular targeting ligand comprising a PNA or another nucleaseresistant oligonucleotide analog that does not activate the degradationof the mRNA by RNase H, such as MOE-mRNA or LNA, having a sequence thatbinds selectively to an uniquely expressed or overexpressed mRNAspecific to the cancer or disease state, having associated therewith apermeation peptide, and a diagnostic imaging detectable amount of atleast one detectably labeled compound.
 2. The diagnostic target-specificimaging probe in accordance with claim 1, wherein the intracellulartargeting ligand, the permeation peptide and the diagnostic imagingdetectable amount of at least one detectably labeled compound areassociated with a functional water dispersible particle syntheticconjugate.
 3. The diagnostic target-specific imaging probe in accordancewith claim 1 wherein the PNA sequence is isolated, purified andcharacterized PNA50 (TGGTGTGCTTTGTGGATG) and the cancer specific mRNA isthe unr mRNA.
 4. The diagnostic target-specific imaging probe inaccordance with claim 2 wherein the PNA sequence is isolated, purifiedand characterized PNA50 (TGGTGTGCTTTGTGGATG).
 5. The diagnostictarget-specific imaging probe in accordance with claim 4 further whereinthe cancer specific mRNA is the unr mRNA.
 6. The diagnostictarget-specific imaging probe in accordance with claim 5 wherein theprobe is compatible with human tissue.
 7. A method of detecting cancercomprising administering an effective amount of an intracellulartargeting ligand comprising a PNA or another nuclease resistantoligonucleotide analog that does not activate the degradation of themRNA by RNase H, having a unr mRNA binding sequence PNA50(TGGTGTGCTTTGTGGATG) or a sequence that reactively and selectively bindsto an uniquely expressed or overexpressed mRNA specific to the cancer, apermeation peptide and a reporter capable of detecting the cancer, suchas a radionuclide, an emission-capable fluorophore, or both aradionuclide and a fluorophore.
 8. The method of detecting cancer inaccordance with claim 7, wherein the intracellular targeting ligand, thepermeation peptide and the reporter capable of detecting the cancer, areassociated with a functional water dispersible particle syntheticconjugate.
 9. The method in accordance with claim 8 wherein the waterdispersible particle synthetic conjugate is effectively administered toa living mammal.
 10. The method in accordance with claim 9 wherein theliving mammal is a living human.
 11. An anticancer composition effectivefor treating human or non-human neoplastic disorder comprising anintracellular targeting ligand comprising a PNA or another nucleaseresistant oligonucleotide analog that does not activate the degradationof the mRNA by RNAseH, having a unr mRNA binding sequence such as PNA50(TGGTGTGCTTTGTGGATG) or any sequence that binds selectively to anuniquely expressed or overexpressed mRNA specific to the cancer ordisease state, a permeation peptide, and an effective amount of at leastone radionuclide with cytotoxic properties, a chemotherapeutic compound,a cytotoxic compound, or a prodrug in the composition, and optionallyfurther comprising a pharmaceutically acceptable carrier such as salinesolution.
 12. The anticancer composition in accordance with claim 11,wherein the intracellular targeting ligand, the permeation peptide andthe effective amount of at least one radiotherapeutic nuclide,chemotherapeutic compound, cytotoxic compound, or prodrug in thecomposition, are associated with a functional water dispersible particlesynthetic conjugate and optionally further comprising a pharmaceuticallyacceptable carrier such as saline solution.
 13. The target-specificanti-cancer composition of claim 4 wherein the PNA sequence is theisolated, purified and characterized PNA50 (TGGTGTGCTTTGTGGATG) and thecancer specific mRNA is the unr mRNA.
 14. The target-specificanti-cancer composition of claim 8, wherein the PNA sequence is theisolated, purified and characterized PNA50 (TGGTGTGCTTTGTGGATG) and thecancer specific mRNA is the unr mRNA.
 15. A method for determiningresponse to anticancer therapy in a living mammal comprisingadministering to a living mammal an imaging probe comprising anintracellular targeting ligand comprising a PNA or another nucleaseresistant oligonucleotide analog that does not activate the degradationof the target mRNA by RNase H, having a unr mRNA binding sequence suchas PNA50 (TGGTGTGCTTTGTGGATG), or any sequence that binds selectively toa unique or overexpressed mRNA specific to the cancer or disease state,a permeation peptide, and a diagnostic imaging detectable amount of atleast one detectably labeled compound at a first selected time,detecting an image of a tissue, administering the imaging probe a secondtime after the anticancer therapy, detecting an image of the sametissue, comparing the diagnostic probe tumor uptake in the two imagesand determining a response based on that comparison.
 16. The method fordetermining response to anticancer therapy in accordance with claim 15,wherein the intracellular targeting ligand, the permeation peptide andthe detectably labeled compound associated with a functional waterdispersible particle synthetic conjugate.
 17. The method for determiningresponse to anticancer therapy of claim 15 wherein the anticancercompound is a candidate chemical for toxicity/lethality to cancer andthe comparison of the images before and after the administration allowfor the determination of the effectiveness of the candidate chemical.18. The method in accordance with claim 17 wherein diagnostic imagingdetectable amount of at least one detectably labeled compound at a firsttime, detecting and acquiring an image of a tissue, administering to themammal a candidate chemical, detecting and acquiring an image of tissue,comparing the detected images and making a determination as to whetherthere has been a prophylactic effect on the progression of the cancer.19. The method for determining response to anticancer therapy inaccordance with claim 18, wherein the anticancer compound is a candidatechemical for toxicity/lethality to cancer and the comparison of theimages before and after the administration allow for the determinationof the effectiveness of the candidate chemical.
 20. The cancer specificdiagnostic, or therapeutic, or diagnostic and therapeutic composition inaccordance with claims 1, 2, 3, 6, 7, and 8, wherein the intracellulartargeting ligand, the permeation peptide, the detectably labeled and thecytotoxic compounds, optionally associated with a functional waterdispersible particle synthetic conjugate, are comprised in apharmaceutical kit containing a suitable pharmaceutically acceptablecarrier, excipient, diluent or saline composition.